Generation of high polyhydroxybutrate producing oilseeds

ABSTRACT

Transgenic oilseed plants, plant material, plant cells, and genetic constructs for synthesis of polyhydroxyalkanoates (“PHA”) are provided. In a preferred embodiment, the transgenic oilseed plants synthesize (poly)3-hydroxybutyrate (“PHB”) in the seed. Genes utilized include phaA, phaB, phaC, all of which are known in the art. The genes can be introduced in the plant, plant tissue, or plant cell using conventional plant molecular biology techniques.

FIELD OF THE INVENTION

The invention is in the field of polymer production in transgenic plants. Methods for generating industrial oilseeds producing high levels of polyhydroxybutyrate (PHB) and industrial oilseeds producing high levels of PHB are described.

BACKGROUND OF THE INVENTION

Production of polyhydroxyalkanoates (PHAs), a family of naturally occurring renewable and biodegradable plastics, in crops has the potential of providing a renewable source of polymers, chemical intermediates and bio-energy from one crop if plant residues remaining after polymer isolation are converted to liquid fuels and/or energy. PHAs can provide an additional revenue stream that would make bioenergy crops more economically viable.

PHAs are a natural component of numerous organisms in multiple ecosystems and accumulate in a wide range of bacteria as a granular storage material when the microbes are faced with an unfavorable growth environment, such as a limitation in an essential nutrient (Madison et al., Microbiol. Mol. Biol. Rev., 1999, 63, 21-53; Suriyamongkol et al., Biotechnol Adv, 2007, 25, 148-175). The monomer unit composition of these polymers is largely dictated by available carbon source as well as the native biochemical pathways present in the organism. Today PHAs are produced industrially from renewable resources in bacterial fermentations providing an alternative to plastics derived from fossil fuels. PHAs possess properties enabling their use in a variety of applications currently served by petroleum-based plastics and are capable of matching or exceeding the performance characteristics of fossil fuel derived plastics with a broad spectrum of properties that can be obtained by varying the monomer composition of homo- and co-polymers, or by manipulating properties such as molecular weight (Sudesh et al., Prog. Polym. Sci., 2000, 25, 1503-1555; Sudesh et al., CLEAN—Soil, Air, Water, 2008, 36, 433-442).

Industrial production of PHAs in crop plants would provide a low cost, renewable source of plastics. Production of PHAs in plants has been an as yet unsolved goal for plant scientists and has previously been demonstrated in a number of crops unsuitable for industrial production or in industrially useful crops at levels to low to be commercially attractive [for review, see (Suriyamongkol et al., Biotechnol Adv, 2007, 25, 148-175); (van Beilen et al., The Plant Journal, 2008, 54, 684-701) and references within] including maize (Poirier et al., 2002, Polyhydroxyalkanoate production in transgenic plants, in Biopolymers, Vol 3a, Steinbuchel, A. (ed), Wiley-VHC Verlag GmbH, pgs 401-435), sugarcane (Purnell et al., Plant Biotechnol. J., 2007, 5, 173-184), switchgrass (Somleva et al., Plant Biotechnol J, 2008, 6, 663-678), flax (Wrobel et al., J. Biotechnol., 2004, 107, 41-54; Wrobel-Kwiatkowsk et al., Biotechnol Prog, 2007, 23, 269-277), cotton (John et al., Proceedings of the National Academy of Sciences of the United States of America, 1996, 93, 12768-12773), alfalfa (Saruul et al., Crop Sci., 2002, 42, 919-927), tobacco (Arai et al., Plant Biotechnol., 2001, 18, 289-293; Bohmert et al., Plant Physiol., 2002, 128, 1282-1290; Lossl et al., Plant Cell Reports, 2003, 21, 891-899; Lössl et al., Plant Cell Physiol, 2005, 46, 1462-1471), potato (Bohmert et al., Plant Physiol., 2002, 128, 1282-1290), and oilseed rape (Valentin et al., Int. J. Biol. Macromol., 1999, 25, 303-306; Slater et al., Nat. Biotechnol., 1999, 17, 1011-1016). Most of the efforts to produce PHAs in plants have focused on production of the homopolymer P3HB or the copolymer poly-3-hydroxybutyrate-co-3-hydroxyvalerate (P3HBV). While there have been some efforts to produce medium chain length PHAs in plants, these studies have yielded barely detectable levels of polymer (Romano et al., Planta, 2005, 220, 455-464; Mittendorf et al., Proceedings of the National Academy of Sciences of the United States of America, 1998, 95, 13397-13402; Poirier et al., Plant Physiol., 1999, 121, 1359-1366; Matsumoto, Journal of Polymers and the Environment, 2006, 14, 369-374; Wang et al., Chinese Science Bulletin, 2005, 50, 1113-1120).

To date, the highest levels of polymer have been obtained when the homopolymer poly-3-hydroxybutyrate (P3HB or PHB) is produced in plastids (Suriyamongkol et al., Biotechnol Adv, 2007, 25, 148-175; van Beilen et al., The Plant Journal, 2008, 54, 684-701; Bohmert et al., Molecular Biology and Biotechnology of Plant Organelles, 2004, 559-585). This is likely due to the high flux of acetyl-CoA, the precursor for PHB in these organelles during fatty acid biosynthesis (Bohmert et al, Molecular Biology and Biotechnology of Plant Organelles, 2004, 559-585). Expression of three genes encoding β-ketothiolase, acetoacetyl CoA reductase, and PHA synthase, allows the conversion of acetyl-CoA within the plastid to PHB. Previous work has reported producing levels of PHB in Brassica napus up to a maximum of 6.7% of seed weight, a level too low for commercial production

SUMMARY OF THE INVENTION

Transgenic oilseed plants, plant material, plant cells, and genetic constructs for synthesis of polyhydroxyalkanoates (“PHA”) are provided. In a preferred embodiment, the transgenic oilseed plants synthesize (poly)3-hydroxybutyrate (“PHB”) in the seed. Host plants, plant tissue, and plant material have been engineered to express genes encoding enzymes in the biosynthetic pathway for PHB production such that polymer precursors in the plastid are polymerized to polymer. Genes utilized include phaA, phaB, phaC, all of which are known in the art. The genes can be introduced in the plant, plant tissue, or plant cell using conventional plant molecular biology techniques.

It is an object of the invention to provide methods and compositions for producing transgenic oilseeds having commercially viable levels of polyhydroxyalkanoates in the seed, for example greater than 7%, 10%, 15%, or 19% polyhydroxyalkanoate or more of the total dry seed weight.

It is another object of the invention to provide oilseeds having increased levels of polyhydroxyalkanoate greater than 7%, 10%, 15%, or 19% polyhydroxyalkanoate or more of the total dry seed weight and having impaired germination relative to non-transgenic oilseeds.

Using a non-traditional screening method to identify transgenic lines than those used in all other reported studies, it has been discovered that very high levels of PHA, for example PHB can be produced in the oilseed but that oilseeds with high levels of PHA fail to germinate or germinate but produce impaired seedlings which do not survive to produce viable fertile plants. The failure to produce viable progeny explains why previous researchers failed to demonstrate that commercial levels of PHA can be produced in transgenic oilseeds. A preferred PHA produced in oilseeds is PHB.

In another embodiment the transgenes encoding PHA biosynthesis genes are expressed in a seed specific manner such that the PHA accumulates in the seed. In this embodiment the level of PHA accumulated is greater than 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% and 19% of the dry weight of the seed.

Methods and compositions for producing hybrid lines are also provided. Hybrid lines can be created by crossing a line containing one or more PHAs, for example PHB genes with a line containing the other gene(s) needed to complete the PHA biosynthetic pathway. Use of lines that possess cytoplasmic male sterility with the appropriate maintainer and restorer lines allows these hybrid lines to be produced efficiently.

In still another embodiment the oilseeds produced by the disclosed methods produce high levels of PHA and are impaired in their ability to germinate and survive to produce viable plants relative to oilseeds containing little or no PHA, for example less than 7% PHA of the dry weight of the seed. Germination can be impaired by 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% relative to oilseeds with less than 7% PHA. Impaired germination provides a built in mechanism for gene containment reducing the risk of unwanted growth of these oilseeds when a different crop is planted on the production fields.

Transgenic plants useful for the invention include dicots or monocots. Preferred host plants are oilseed plants, but are not limited to members of the Brassica family including B. napus, B. rapa, B. carinata and B. juncea. Additional preferred host plants include industrial oilseeds such as Camelina sativa, Crambe, jatropha, and castor. Other preferred host plants include Arabidopsis thaliana, Calendula, Cuphea, maize, soybean, cottonseed, sunflower, palm, coconut, safflower, peanut, mustards including Sinapis alba, and tobacco.

Other embodiments provide plant material and plant parts of the transgenic plants including seeds, flowers, stems, and leaves. The oilseeds can be used for the extraction of PHA biopolymer or as a source of PHA biopolymer based chemical intermediates. The residual parts of the seed can be used as meal for animal feed or steam and power generation and a source of vegetable oil for industrial oelochemicals or biofuel.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram describing a strategy for creating hybrid seeds using cytoplasmic male sterility.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Unless otherwise indicated, the disclosure encompasses all conventional techniques of plant breeding, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd edition (2001); Current Protocols In Molecular Biology [(F. M. Ausubel, et al. eds., (1987)]; Plant Breeding Principles and Prospects (Plant Breeding, Vol 1) M. D. Hayward, N. O. Bosemark, I. Romagosa; Chapman & Hall, (1993.); Coligan, Dunn, Ploegh, Speicher and Wingfeld, eds. (1995) Current Protocols in Protein Science (John Wiley & Sons, Inc.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)].

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Lewin, Genes VII, published by Oxford University Press, 2000; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Wiley-Interscience., 1999; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology, a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995; Ausubel et al. (1987) Current Protocols in Molecular Biology, Green Publishing; Sambrook and Russell. (2001) Molecular Cloning: A Laboratory Manual 3rd. edition.

A number of terms used herein are defined and clarified in the following section.

The term PHB refers to polyhydroxybutyrate and is used interchangeably with the term PHA which refers to polyhydroxyalkanoate.

The tend PHB also encompasses copolymers of hydroxybutyrate with other hydroxyacid monomers.

The term “PHA copolymer” refers to a polymer composed of at least two different hydroxyalkanoic acid monomers.

The term “PHA homopolymer” refers to a polymer that is composed of a single hydroxyalkanoic acid monomer.

As used herein, a “vector” is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. The vectors can be expression vectors.

As used herein, an “expression vector” is a vector that includes one or more expression control sequences

As used herein, an “expression control sequence” is a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence. Control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, a ribosome binding site, and the like. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

As used herein, “operably linked” means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest.

As used herein, “transformed” and “transfected” encompass the introduction of a nucleic acid into a cell by a number of techniques known in the art.

“Plasmids” are designated by a lower case “p” preceded and/or followed by capital letters and/or numbers.

As used herein the term “heterologous” means from another host. The other host can be the same or different species.

The term “cell” refers to a membrane-bound biological unit capable of replication or division.

The term “construct” refers to a recombinant genetic molecule including one or more isolated polynucleotide sequences.

Genetic constructs used for transgene expression in a host organism comprise in the 5′-3′ direction, a promoter sequence; a nucleic acid sequence encoding the desired transgene product; and a termination sequence. The open reading frame may be orientated in either a sense or anti-sense direction. The construct may also comprise selectable marker gene(s) and other regulatory elements for expression.

The term “plant” is used in it broadest sense. It includes, but is not limited to, any species of woody, ornamental or decorative, crop or cereal, fruit or vegetable plant, and photosynthetic green algae (e.g., Chlamydomonas reinhardtii). It also refers to a plurality of plant cells that are largely differentiated into a structure that is present at any stage of a plant's development. Such structures include, but are not limited to, a fruit, shoot, stem, leaf, flower petal, etc. The term “plant tissue” includes differentiated and undifferentiated tissues of plants including those present in roots, shoots, leaves, pollen, seeds and tumors, as well as cells in culture (e.g., single cells, protoplasts, embryos, callus, etc.). Plant tissue may be in planta, in organ culture, tissue culture, or cell culture. The term “plant part” as used herein refers to a plant structure, a plant organ, or a plant tissue.

A non-naturally occurring plant refers to a plant that does not occur in nature without human intervention. Non-naturally occurring plants include transgenic plants and plants produced by non-transgenic means such as plant breeding.

The term “plant cell” refers to a structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, a plant tissue, a plant organ, or a whole plant.

The term “plant cell culture” refers to cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development.

The term “plant material” refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.

A “plant organ” refers to a distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower bud, or embryo.

“Plant tissue” refers to a group of plant cells organized into a structural and functional unit. Any tissue of a plant, whether in a plant or in culture, is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture and any groups of plant cells organized into structural and/or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.

“Seed germination” refers to growth of an embryonic plant contained within a seed resulting in the formation and emergence of a seedling.

“Cotyledon” refers to the embryonic first leaves of a seedling.

“Early plantlet development” refers to growth of the cotyledon containing seedling to form a plantlet.

II. Transgenic Plants

Transgenic plants have been developed that produce increased levels of biopolymers such as polyhydroxyalkanoates (PHAs) in seeds. Methods and constructs for engineering plants for seed specific production of PHA, in particular PHB, are described. One embodiment provides transgenic plants for the direct, large scale production of PHAs in crop plants or in energy crops where a plant by-product, such as oil, can be used for production of energy. Proof of concept studies for polyhydroxybutyrate (PHB) synthesis in canola (Valentin et al., Int. J. Biol. Macromol., 1999, 25, 303-306; Houmiel et al., Planta, 1999, 209, 547-550; Slater et al., Nat. Biotechnol., 1999, 17, 1011-1016.) have been reported. There have been instances where high level PHB production in the chloroplasts of plants has led to decreases in total plant growth (Bohmert et al., Molecular Biology and Biotechnology of Plant Organelles, 2004, 559-585; Bohmert et al., Planta, 2000, 211, 841-845) for unidentified reasons. There have been several studies that have attempted to alleviate this problem by inducible expression of enzymes (Bohmert et al., Plant Physiol., 2002, 128, 1282-1290; Lössl et al., Plant Cell Physiol, 2005, 46, 1462-1471; Kourtz et al., Transgenic Res, 2007, 16, 759-769).

Transgenic oilseeds comprising at least about 8% dry weight PHA are provided. In one embodiment we provide transgenic oilseeds having at least 10% PHA dry weight and which are impaired in germination and plant survival.

A. Genetic Constructs for Transformation

Suitable genetic constructs include expression cassettes for enzymes for production of polyhydroxyalkanoates, in particular from the polyhydroxybutyrate biosynthetic pathway. In one embodiment, the construct contains operatively linked in the 5′ to 3′ direction, a seed specific promoter that directs transcription of a nucleic acid sequence in the nucleus; a nucleic acid sequence encoding one of the PHB biosynthetic enzymes; and a 3′ polyadenylation signal that increases levels of expression of transgenes. In one embodiment, enzymes for formation of polymer precursors are targeted to the plastid using appropriate plastid-targeting signals. In another embodiment, the PHA pathway is expressed directly from the plastid genome using appropriate plastidial promoters and regulatory sequences.

DNA constructs useful in the methods described herein include transformation vectors capable of introducing transgenes into plants. As used herein, “transgenic” refers to an organism in which a nucleic acid fragment containing a heterologous nucleotide sequence has been introduced. The transgenes in the transgenic organism are preferably stable and inheritable. The heterologous nucleic acid fragment may or may not be integrated into the host genome.

Several plant transformation vector options are available, including those described in “Gene Transfer to Plants” (Potrykus, et al., eds.) Springer-Verlag Berlin Heidelberg New York (1995); “Transgenic Plants: A Production System for Industrial and Pharmaceutical Proteins” (Owen, et al., eds.) John Wiley & Sons Ltd. England (1996); and “Methods in Plant Molecular Biology: A Laboratory Course Manual” (Maliga, et al. eds.) Cold Spring Laboratory Press, New York (1995). Plant transformation vectors generally include one or more coding sequences of interest under the transcriptional control of 5′ and 3′ regulatory sequences, including a promoter, a transcription termination and/or polyadenylation signal, and a selectable or screenable marker gene. For the expression of two or more polypeptides from a single transcript, additional RNA processing signals and ribozyme sequences can be engineered into the construct (U.S. Pat. No. 5,519,164). This approach has the advantage of locating multiple transgenes in a single locus, which is advantageous in subsequent plant breeding efforts.

Engineered minichromosomes can also be used to express one or more genes in plant cells. Cloned telomeric repeats introduced into cells may truncate the distal portion of a chromosome by the formation of a new telomere at the integration site. Using this method, a vector for gene transfer can be prepared by trimming off the arms of a natural plant chromosome and adding an insertion site for large inserts (Yu et al., Proc Natl Acad Sci USA, 2006, 103, 17331-6; Yu et al., Proc Natl Acad Sci USA, 2007, 104, 8924-9). The utility of engineered minichromosome platforms has been shown using Cre/lox and FRT/FLP site-specific recombination systems on a maize minichromosome where the ability to undergo recombination was demonstrated (Yu et al., Proc Natl Acad Sci USA, 2006, 103, 17331-6; Yu et al., Proc Natl Acad Sci USA, 2007, 104, 8924-9). Such technologies could be applied to minichromosomes, for example, to add genes to an engineered plant. Site specific recombination systems have also been demonstrated to be valuable tools for marker gene removal (Kerbach, S. et al., Theor Appl Genet, 2005, 111, 1608-1616), gene targeting (Chawla, R. et al., Plant Biotechnol J, 2006, 4, 209-218; Choi, S. et al., Nucleic Acids Res, 2000, 28, E19; Srivastava, V, & Ow, D W, Plant Mol Biol, 2001, 46, 561-566; Lyznik, L A, et al., Nucleic Acids Res, 1993, 21, 969-975), and gene conversion (Djukanovic, V, et al., Plant Biotechnol J, 2006, 4, 345-357).

An alternative approach to chromosome engineering in plants involves in vivo assembly of autonomous plant minichromosomes (Carlson et al., PLoS Genet, 2007, 3, 1965-74). Plant cells can be transformed with centromeric sequences and screened for plants that have assembled autonomous chromosomes de novo. Useful constructs combine a selectable marker gene with genomic DNA fragments containing centromeric satellite and retroelement sequences and/or other repeats.

Another approach is Engineered Trait Loci (“ETL”) technology (U.S. Pat. No. 6,077,697 to Hadlaczky et al.; US Patent Application 2006/0143732). This system targets DNA to a heterochromatic region of plant chromosomes, such as the pericentric heterochromatin, in the short arm of acrocentric chromosomes. Targeting sequences may include ribosomal DNA (rDNA) or lambda phage DNA. The pericentric rDNA region supports stable insertion, low recombination, and high levels of gene expression. This technology is also useful for stacking of multiple traits in a plant (US Patent Application 2006/0246586, 2010/0186117 and PCT WO 2010/037209).

Zinc-finger nucleases (ZFNs) are also useful in that they allow double strand DNA cleavage at specific sites in plant chromosomes such that targeted gene insertion or deletion can be performed (Shukla et al., Nature, 2009; Townsend et al., Nature, 2009).

For direct expression of transgenes from the plastid genome, a vector to transform the plant plastid chromosome by homologous recombination (as described in U.S. Pat. No. 5,545,818 to McBride et al.) is used in which case it is possible to take advantage of the prokaryotic nature of the plastid genome and insert a number of transgenes as an operon. WO 2010/061186 describes an alternative method for introducing genes into the plastid chromosome using an adapted endogenous cellular process for the transfer of RNAs from the cytoplasm to the plastid where they are incorporated by homologous recombination. This plastid transformation procedure is also suitable for practicing the disclosed compositions and methods.

A transgene may be constructed to encode a multifunctional enzyme through gene fusion techniques in which the coding sequences of different genes are fused with or without linker sequences to obtain a single gene encoding a single protein with the activities of the individual genes. Transgenes encoding a bifunctional protein containing thiolase and reductase activities (Kourtz, L., K. et al. (2005), Plant Biotechnol. 3: 435-447) and a trifunctional protein having each of the three enzyme activities required for PHB expression in plants (Mullaney and Rehm (2010), Journal of Biotechnology 147: 31-36) have been described. Such synthetic fusion gene/enzyme combinations can be further optimized using molecular evolution technologies.

A transgene may be constructed to encode a series of enzyme activities separated by intein sequences such that on expression, two or more enzyme activities are expressed from a single promoter as described by Snell in U.S. Pat. No. 7,026,526 to Metabolix, Inc.

1. Genes Involved in Polyhydroxyalkanoate Synthesis

In a preferred embodiment, the products of the transgenes are enzymes and other factors required for production of a biopolymer, such as a polyhydroxyalkanoate (PHA).

For PHA production, transgenes encode enzymes such as beta-ketothiolase, acetoacetyl-CoA reductase, PHB (“short chain”) synthase, PHA (“long chain”) synthase, threonine dehydratase, dehydratases such as 3-OH acyl ACP, isomerases such as Δ 3-cis, Δ 2-trans isomerase, propionyl-CoA synthetase, hydroxyacyl-CoA synthetase, hydroxyacyl-CoA transferase, R-3-hydroxyacyl-ACP:CoA transferase, thioesterase, fatty acid synthesis enzymes and fatty acid beta-oxidation enzymes. Useful genes are well known in the art, and are disclosed for example by Snell and Peoples Metab. Eng. 4: 29-40 (2002); Bohmert et. al. in Molecular Biology and Biotechnology of Plant Organelles. H. Daniell, C. D. Chase Eds., Kluwer Academic Publishers, Netherlands, 2004, pp. 559-585; (Suriyamongkol et al., Biotechnol Adv, 2007, 25, 148-175; van Beilen et al., The Plant Journal, 2008, 54, 684-701).

PHA Synthases

Examples of PHA synthases include a synthase with medium chain length substrate specificity, such as phaC1 from Pseudomonas oleovorans (WO 91/000917; Huisman, et al. J. Biol. Chem. 266, 2191-2198 (1991)) or Pseudomonas aeruginosa (Timm, A. & Steinbuchel, A. Eur. J. Biochem. 209: 15-30 (1992)), the synthase from Alcaligenes eutrophus with short chain length specificity (Peoples, O. P. & Sinskey, A. J. J. Biol. Chem. 264:15298-15303 (1989)), or a two subunit synthase such as the synthase from Thiocapsa pfennigii encoded by phaE and phaC (U.S. Pat. No. 6,011,144). Other useful PHA synthase genes have been isolated from, for example, Alcaligenes latus (Accession ALU47026), Burkholderia sp. (Accession AF153086), Aeromonas caviae (Fukui & Doi, J. Bacteriol. 179: 4821-30 (1997)), Acinetobacter sp. strain RA3849 (Accession L37761), Rhodospirillum rubrum (U.S. Pat. No. 5,849,894), Rhodococcus ruber (Pieper & Steinbuechel, FEMS Microbiol. Lett. 96(1): 73-80 (1992)), and Nocardia corallina (Hall et. al., Can. J. Microbiol. 44: 687-91 (1998)), Arthrospira sp. PCC 8005 (Accessions ZP_(—)07166315 and ZP_(—)07166316), Cyanothece sp. PCC 7425 (Accessions ACL46371 and ACL46370) and Synechocystis sp. PCC6803 (Accession BAA17430; Hein et al. (1998), Archives of Microbiology 170: 162-170).

PHA synthases with broad substrate specificity useful for producing copolymers of 3-hydroxybutyrate and longer chain length (from 6 to 14 carbon atoms) hydroxyacids have also been isolated from Pseudomonas sp. A33 (Appl. Microbiol. Biotechnol. 42: 901-909 (1995)) and Pseudomonas sp. 61-3 (Accession AB014757; Kato, et al. Appl. Microbiol. Biotechnol. 45: 363-370 (1996)).

A range of PHA synthase genes and genes encoding additional metabolic steps useful in PHA biosynthesis are described by Madison and Huisman. Microbiology and Molecular biology Reviews 63:21-53 (1999)) and Suriyamongkol et al. (Suriyamongkol et al., Biotechnol Adv, 2007, 25, 148-175).

Hydratase and Dehydrogenase

An alpha subunit of beta-oxidation multienzyme complex pertains to a multifunctional enzyme that minimally possesses hydratase and dehydrogenase activities. The subunit may also possess epimerase and A 3-cis, A 2-trans isomerase activities. Examples of alpha subunits of the beta-oxidation multienzyme complex are FadB from E. coli (DiRusso, C. C. J. Bacteriol. 1990, 172, 6459-6468), FaoA from Pseudomonas fragi (Sato, S., Hayashi, et al. J. Biochem. 1992, 111, 8-15), and the E. coli open reading frame 1714 that contains homology to multifunctional α subunits of the β-oxidation complex (Genbank Accession #1788682). A β subunit of the β-oxidation complex refers to a polypeptide capable of forming a multifunctional enzyme complex with its partner α subunit. The β subunit possesses thiolase activity. Examples of β subunits are FadA from E. coli (DiRusso, C. C. J. Bacteriol. 172: 6459-6468 (1990)), FaoB from Pseudomonas fragi (Sato, S., Hayashi, M., Imamura, S., Ozeki, Y., Kawaguchi, A. J. Biochem. 111: 8-15 (1992)), and the E. coli open reading frame f436 that contains homology to α subunits of the β-oxidation complex (Genbank Accession # AE000322; gene b2342).

Reductases

The transgene can encode a reductase. A reductase refers to an enzyme that can reduce β-ketoacyl CoAs to R-3-OH-acyl CoAs, such as the NADH dependent reductase from Chromatium vinosum (Liebergesell, M., & Steinbuchel, A. Eur. J. Biochem. 209: 135-150 (1992)), the NADPH dependent reductase from Alcaligenes eutrophus (Accession J04987, Peoples, O. P. & Sinskey, A. J. J. Biol. Chem. 264: 15293-15297 (1989))), the NADPH reductase from Zoogloea ramigera (Accession P23238; Peoples, O. P. & Sinskey, A. J. Molecular Microbiology 3: 349-357 (1989)) or the NADPH reductase from Bacillus megaterium (U.S. Pat. No. 6,835,820), Alcaligenes latus (Accession ALU47026), Rhizobium meliloti (Accession RMU17226), Paracoccus denitrificans (Accession D49362), Burkholderia sp. (Accession AF153086), Pseudomonas sp. strain 61-3 (Accession AB014757), Acinetobacter sp. strain RA3849 (Accession L37761), P. denitrificans, (Accession P50204), and Synechocystis sp. Strain PCC6803 (Taroncher-Oldenburg et al., (2000), Appl. Environ. Microbiol. 66: 4440-4448).

Thiolases

The transgene can encode a thiolase. A beta-ketothiolase refers to an enzyme that can catalyze the conversion of acetyl CoA and an acyl CoA to a β-ketoacyl CoA, a reaction that is reversible. An example of such thiolases are PhaA from Alcaligenes eutropus (Accession J04987, Peoples, O. P. & Sinskey, A. J. J. Biol. Chem. 264: 15293-15297 (1989)), BktB from Alcaligenes eutrophus (Slater et al. J Bacteriol. 180(8):1979-87 (1998)) and thiolases from the following Rhizobium meliloti (Accession RMU17226), Z. ramigera (Accession P07097), Paracoccus denitrificans (Accession D49362), Burkholderia sp. (Accession AF153086), Alcaligenes latus (Accession ALU47026), Allochromatium vinosum (Accession P45369), Thiocystis violacea (Accession P45363); Pseudomonas sp. strain 61-3 (Accession AB014757), Acinetobacter sp. strain RA3849 (Accession L37761) and Synechocystis sp. Strain PCC6803 (Taroncher-Oldenburg et al., (2000), Appl. Environ. Microbiol. 66: 4440-4448).

Oxidases

An acyl CoA oxidase refers to an enzyme capable of converting saturated acyl CoAs to Δ 2 unsaturated acyl CoAs. Examples of acyl CoA oxidases are POX1 from Saccharomyces cerevisiae (Dmochowska, et al. Gene, 1990, 88, 247-252) and ACX1 from Arabidopsis thaliana (Genbank Accession # AF057044).

Catalases

The transgene can also encode a catalase. A catalase refers to an enzyme capable of converting hydrogen peroxide to hydrogen and oxygen. Examples of catalases are KatB from Pseudomonas aeruginosa (Brown, et al.): Bacterial. 177: 6536-6544 (1995)) and KatG from E. coli (Triggs-Raine, B. L. & Loewen, P. C. Gene 52: 121-128 (1987)).

2. Promoters

Plant promoters can be selected to control the expression of the transgene in different plant tissues or organelles for all of which methods are known to those skilled in the art (Gasser & Fraley, Science 244:1293-99 (1989)). In one embodiment, promoters are selected from those of eukaryotic or synthetic origin that are known to yield high levels of expression in plant and algae cytosol. In another embodiment, promoters are selected from those of plant or prokaryotic origin that are known to yield high expression in plastids. In certain embodiments the promoters are inducible. Inducible plant promoters are known in the art.

Suitable constitutive promoters for nuclear-encoded expression include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in U.S. Pat. No. 6,072,050; the core CAMV 355 promoter, (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); and ALS promoter (U.S. Pat. No. 5,659,026). Other constitutive promoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142.

“Tissue-preferred” promoters can be used to target a gene expression within a particular tissue such as seed, leaf or root tissue. Tissue-preferred promoters include Yamamoto et al. (1997) Plant J 12(2)255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al (1997) Mol. Gen. Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al (1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505.

“Seed-preferred” promoters include both “seed-specific” promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as “seed-germinating” promoters (those promoters active during seed germination). See Thompson et al. (1989) BioEssays 10:108. Such seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa zein); milps (myo-inositol-1-phosphate synthase); and ce1A (cellulose synthase). Gama-zein is a preferred endosperm-specific promoter. Glob-1 is a preferred embryo-specific promoter. For dicots, seed-specific promoters include, but are not limited to, bean β-phaseolin, napin β-conglycinin, soybean lectin, cruciferin, oleosin, the Lesquerella hydroxylase promoter, and the like. For monocots, seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. Additional seed specific promoters useful for practicing this invention are described in the Examples disclosed herein.

Leaf-specific promoters are known in the art. See, for example, Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

Root-preferred promoters are known and may be selected from the many available from the literature or isolated de nova from various compatible species. See, for example, Hire et al. (1992) Plant Mol. Biol. 20(2): 207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-specific control element in the GRP 1.8 gene of French bean); Sanger et al. (1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao et al. (1991) Plant Cell 3(1):1 1′-22 (full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean). See also U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179.

Plastid specific promoters include the PrbcL promoter [Allison L. A. et al., EMBO 15: 2802-2809 (1996); Shiina T. et al., Plant Cell 10: 1713-1722 (1998)]; the PpsbA promoter [Agrawal G K, et al., Nucleic Acids Research 29: 1835-1843 (2001)]; the Prrn 16 promoter [Svab Z & Maliga P., Proc. Natl. Acad. Sci. USA 90: 913-917 (1993), Allison L A et al., EMBO 15: 2802-2809 (1996)]; the PaccD promoter (WO97/06250; Hajdukiewicz P T J et al., EMBO J. 16: 4041-4048 (1997)).

Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize 1n2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1 a promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. Proc. Natl. Acad. Sci. USA 88:10421-10425 (1991) and McNellis et al. Plant J 14(2):247-257 (1998)) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. Mol. Gen. Genet. 227:229-237 (1991), and U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by reference in their entirety.

In one embodiment, coordinated expression of the three transgenes, phaA, phaB, and phaC, necessary for conversion of acetyl-CoA to PHB is controlled by a seed specific promoter, such as the soybean oleosin promoter (Rowley et al., Biochim Biophys Acta, 1997, 1345, 1-4) or the promoter from the lesquerlla hydroxylase gene (U.S. Pat. No. 6,437,220 B1). In another embodiment, coordinated expression of the three transgenes, phaA, phaB, and phaC, necessary for conversion of acetyl-CoA to PHB is controlled by a promoter active primarily in the biomass plant, such as the maize chlorophyll A/B binding protein promoter (Sullivan et al., Mol. Gen. Genet., 1989, 215, 431-40). It has been previously shown that plants transformed with multi-gene constructs produced higher levels of polymer than plants obtained from crossing single transgene lines (Valentin et al., Int. J. Biol. Macromol., 1999, 25, 303-306; Bohmert et al., Planta, 2000, 211, 841-845).

In one embodiment, the final molecular weight of the polymer produced is controlled by the choice of promoter for expression of the PHA synthase gene. As described in U.S. Pat. No. 5,811,272, high PHA synthase activity will lower polymer molecular weight and low PHA synthase activity will increase polymer molecular weight. In another embodiment, a strong promoter is used for expression of the genes encoding plastid-targeted monomer producing enzymes while a weaker promoter is used to control expression of synthase.

3. Transcription Termination Sequences

At the extreme 3′ end of the transcript of the transgene, a polyadenylation signal can be engineered. A polyadenylation signal refers to any sequence that can result in polyadenylation of the mRNA in the nucleus prior to export of the mRNA to the cytosol, such as the 3′ region of nopaline synthase (Bevan, M., Barnes, W. M., Chilton, M. D. Nucleic Acids Res. 1983, 11, 369-385).

4. Selectable Markers

Genetic constructs may encode a selectable marker to enable selection of plastid transformation events. There are many methods that have been described for the selection of transformed plants [for review see (Miki et al., Journal of Biotechnology, 2004, 107, 193-232) and references incorporated within]. Selectable marker genes that have been used extensively in plants include the neomycin phosphotransferase gene nptII (U.S. Pat. No. 5,034,322, U.S. Pat. No. 5,530,196), hygromycin resistance gene (U.S. Pat. No. 5,668,298), the bar gene encoding resistance to phosphinothricin (U.S. Pat. No. 5,276,268), the expression of aminoglycoside 3″-adenyltransferase (aadA) to confer spectinomycin resistance (U.S. Pat. No. 5,073,675), the use of inhibition resistant 5-enolpyruvyl-3-phosphoshikimate synthetase (U.S. Pat. No. 4,535,060) and methods for producing glyphosate tolerant plants (U.S. Pat. No. 5,463,175; U.S. Pat. No. 7,045,684). Methods of plant selection that do not use antibiotics or herbicides as a selective agent have been previously described and include expression of glucosamine-6-phosphate deaminase to inactive glucosamine in plant selection medium (U.S. Pat. No. 6,444,878) and a positive/negative system that utilizes D-amino acids (Erikson et al., Nat Biotechnol, 2004, 22, 455-8). European Patent Publication No. EP 0 530 129 A1 describes a positive selection system which enables the transformed plants to outgrow the non-transformed lines by expressing a transgene encoding an enzyme that activates an inactive compound added to the growth media. U.S. Pat. No. 5,767,378 describes the use of mannose or xylose for the positive selection of transgenic plants. Methods for positive selection using sorbitol dehydrogenase to convert sorbitol to fructose for plant growth have also been described (WO 2010/102293). Screenable marker genes include the beta-glucuronidase gene (Jefferson et al., 1987, EMBO J. 6: 3901-3907; U.S. Pat. No. 5,268,463) and native or modified green fluorescent protein gene (Cubitt et al., 1995, Trends Biochem. Sci. 20: 448-455; Pan et al., 1996, Plant Physiol. 112: 893-900).

Transformation events can also be selected through visualization of fluorescent proteins such as the fluorescent proteins from the nonbioluminescent Anthozoa species which include DsRed, a red fluorescent protein from the Discosoma genus of coral (Matz et al. (1999), Nat Biotechnol 17: 969-73). An improved version of the DsRed protein has been developed (Bevis and Glick (2002), Nat Biotech 20: 83-87) for reducing aggregation of the protein. Visual selection can also be performed with the yellow fluorescent proteins (YFP) including the variant with accelerated maturation of the signal (Nagai, T. et al. (2002), Nat Biotech 20: 87-90), the blue fluorescent protein, the cyan fluorescent protein, and the green fluorescent protein (Sheen et al. (1995), Plant J 8: 777-84; Davis and Vierstra (1998), Plant Molecular Biology 36: 521-528). A summary of fluorescent proteins can be found in Tzfira et al. (Tzfira et al. (2005), Plant Molecular Biology 57: 503-516) and Verkhusha and Lukyanov (Verkhusha, V. V. and K. A. Lukyanov (2004), Nat Biotech 22: 289-296) whose references are incorporated in entirety. Improved versions of many of the fluorescent proteins have been made for various applications. Use of the improved versions of these proteins or the use of combinations of these proteins for selection of transformants will be obvious to those skilled in the art. It is also practical to simply analyze progeny from transformation events for the presence of the PHB thereby avoiding the use of any selectable marker.

For plastid transformation constructs, a preferred selectable marker is the spectinomycin-resistant allele of the plastid 16S ribosomal RNA gene (Staub J M, Maliga P, Plant Cell 4: 39-45 (1992); Svab Z, Hajdukiewicz P, Maliga P, Proc. Natl., Acad. Sci. USA 87: 8526-8530 (1990)). Selectable markers that have since been successfully used in plastid transformation include the bacterial aadA gene that encodes aminoglycoside adenyltransferase (AadA) conferring spectinomycin and streptomycin resistance (Svab et al., Proc, Natl. Acad. Sci. USA, 1993, 90, 913-917), nptII that encodes aminoglycoside phosphotransferase for selection on kanamycin (Carrer H, Hockenberry T N, Svab Z, Maliga P., Mol. Gen. Genet. 241: 49-56 (1993); Lutz K A, et al., Plant J. 37: 906-913 (2004); Lutz K A, et al., Plant Physiol. 145: 1201-1210 (2007)), aphA6, another aminoglycoside phosphotransferase (Huang F—C, et al, Mol. Genet. Genomics 268: 19-27 (2002)), and chloramphenicol acetyltransferase (Li, W., et al. (2010), Plant Mol Biol, DOI 10.1007/s11103-010-9678-4). Another selection scheme has been reported that uses a chimeric betaine aldehyde dehydrogenase gene (BADH) capable of converting toxic betaine aldehyde to nontoxic glycine betaine (Daniell H, et al., Curr. Genet. 39: 109-116 (2001)).

5. Plastid Targeting Signals

Plastid targeting sequences are known in the art and include the chloroplast small subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco) (de Castro Silva Filho et al, Plant Mal. Biol. 30:769-780 (1996); Schnell et J. Biol. Chem. 266(5):3335-3342 (1991)); 5-(enolpyruvyl)shikimate-3-phosphate synthase (EPSPS) (Archer et al. J. Bioenerg. Biomemb. 22(6):789-810 (1990)); tryptophan synthase (Zhao et al. J. Biol. Chem. 270(11):6081-6087 (1995)); plastocyanin (Lawrence et al. J. Biol. Chem. 272(33):20357-20363 (1997)); chorismate synthase (Schmidt et al. J. Biol. Chem. 268(36):27447-27457 (1993)); and the light harvesting chlorophyll a/b binding protein (LHBP) (Lamppa et al. J. Biol. Chem. 263:14996-14999 (1988)). See also Von Heijne et al. Plant Mol. Biol. Rep. 9:104-126 (1991); Clark et al. J. Biol. Chem. 264:17544-17550 (1989); Della-Cioppa et al. Plant Physiol. 84:965-968 (1987); Romer et al. Biochem. Biophys. Res. Commun. 196:1414-1421 (1993); and Shah et al. Science 233:478-481 (1986). Alternative plastid targeting signals have also been described in the following: US 2008/0263728; Miras, S. et al. (2002), J Biol Chem 277(49): 47770-8; Miras, S. et al. (2007), J Biol Chem 282: 29482-29492.

B. Exemplary Host Plants

Plants transformed in accordance with the present disclosure may be monocots or dicots. The transformation of suitable agronomic plant hosts using vectors for nuclear transformation or direct plastid transformation can be accomplished with a variety of methods and plant tissues. Representative plants useful in the methods disclosed herein include the Brassica family including B. napus, B. rapa, B. carinata and B. juncea; industrial oilseeds such as Camelina sativa, Crambe, jatropha, castor; Calendula, Cuphea, Arabidopsis thaliana; maize; soybean; cottonseed; sunflower; palm; coconut; safflower; peanut; mustards including Sinapis alba; sugarcane flax and tobacco, also are useful with the methods disclosed herein. Representative tissues for transformation using these vectors include protoplasts, cells, callus tissue, leaf discs, pollen, and meristems.

C. Methods of Plant Transformation

Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-mediated transformation (Townsend et al., U.S. Pat. No. 5,563,055; Zhao et al. WO US98/01268), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for example, Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al. (1995) Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); and McCabe et al. Biotechnology 6:923-926 (1988)). Also see Weissinger et al. Ann. Rev. Genet. 22:421-477 (1988); Sanford et al, Particulate Science and Technology 5:27-37 (1987) (onion); Christou et al. Plant Physiol. 87:671-674 (1988) (soybean); McCabe et al. (1988) BioTechnology 6:923-926 (soybean); Finer and McMullen In Vitro Cell Dev. Biol. 27P:175-182 (1991) (soybean); Singh et al. Theor. Appl. Genet. 96:319-324 (1998) (soybean); Dafta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. Proc. Natl. Acad. Sci. USA 85:4305-4309 (1988) (maize); Klein et al. Biotechnology 6:559-563 (1988) (maize); Tomes, U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and 5,324,646; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin) (maize); Klein et al. Plant Physiol. 91:440-444 (1988) (maize); Fromm et al. Biotechnology 8:833-839 (1990) (maize); Hooykaas-Van Slogteren et al. Nature 311:763-764 (1984); Bowen et al., U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. Proc. Natl. Acad. Sci. USA 84:5345-5349 (1987) (Liliaceae); De Wet et al. in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp. 197-209 (1985) (pollen); Kaeppler et al. Plant Cell Reports 9:415-418 (1990) and Kaeppler et al. Theor. Appl. Genet. 84:560-566 (1992) (whisker-mediated transformation); D'Halluin et al. Plant Cell 4:1495-1505 (1992) (electroporation); Li et al. Plant Cell Reports 12:250-255 (1993) and Christou and Ford Annals of Botany 75:407-413 (1995) (rice); Osjoda et al. Nature Biotechnology 14:745-750 (1996) (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference in their entirety. References for protoplast transformation and/or gene gun for Agrisoma technology are described in WO 2010/037209. Methods for transforming plant protoplasts are available including transformation using polyethylene glycol (PEG), electroporation, and calcium phosphate precipitation (see for example Potrykus et al., 1985, Mol. Gen. Genet., 199, 183-188; Potrykus et al., 1985, Plant Molecular Biology Reporter, 3, 117-128), Methods for plant regeneration from protoplasts have also been described [Evans et al., in Handbook of Plant Cell Culture, Vol 1, (Macmillan Publishing Co., New York, 1983); Vasil, 1K in Cell Culture and Somatic Cell Genetics (Academic, Orlando, 1984)].

Methods for transformation of plastids such as chloroplasts are known in the art. See, for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga (1993) EMBO J. 12:601-606. The method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation may be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase (McBride et al., Proc. Natl. Acad. Sci. USA, 1994, 91:7301-7305) or by use of an integrase, such as the phiC31 phage site-specific integrase, to target the gene insertion to a previously inserted phage attachment site (Lutz et al., Plant J, 2004, 37, 906-13). Plastid transformation vectors can be designed such that the transgenes are expressed from a promoter sequence that has been inserted with the transgene during the plastid transformation process or, alternatively, from an endogenous plastidial promoter such that an extension of an existing plastidial operon is achieved (Herz et al., Transgenic Research, 2005, 14, 969-982). An alternative method for plastid transformation as described in WO 2010/061186 wherein RNA produced in the nucleus of a plant cell can be targeted to the plastid genome can also be used to practice the disclosed invention. Inducible gene expression from the plastid genome using a synthetic riboswitch has also been reported (Verhounig et al. (2010), Proc Natl Acad Sci USA 107: 6204-6209). Methods for designing plastid transformation vectors are described by Lutz et al. (Lutz et al., Plant Physiol, 2007, 145, 1201-10).

Recombinase technologies which are useful for producing the disclosed transgenic plants include the cre-lox, FLP/FRT and Gin systems. Methods by which these technologies can be used for the purpose described herein are described for example in (U.S. Pat. No. 5,527,695; Dale And Ow, 1991, Proc. Natl. Acad. Sci. USA 88: 10558-10562; Medberry et al., 1995, Nucleic Acids Res. 23: 485-490).

D. Methods for Reproducing Transgenic Plants

Following transformation by any one of the methods described above, the following procedures can be used to obtain a transformed plant expressing the transgenes: select the plant cells that have been transformed on a selective medium; regenerate the plant cells that have been transformed to produce differentiated plants; select transformed plants expressing the transgene producing the desired level of desired polypeptide(s) in the desired tissue and cellular location.

In plastid transformation procedures, further rounds of regeneration of plants from explants of a transformed plant or tissue can be performed to increase the number of transgenic plastids such that the transformed plant reaches a state of homoplasmy (all plastids contain uniform plastomes containing transgene insert).

The cells that have been transformed may be grown into plants in accordance with conventional techniques. See, for example, McCormick et al, Plant Cell Reports 5:81-84 (1986). These plants may then be grown, and either pollinated with the same transformed variety or different varieties, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that constitutive expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure constitutive expression of the desired phenotypic characteristic has been achieved.

In some scenarios, it may be advantageous to insert a multi-gene pathway into the plant by crossing of lines containing portions of the pathway to produce hybrid plants in which the entire pathway has been reconstructed. This is especially the case when high levels of product in a seed compromises the ability of the seed to germinate or the resulting seedling to survive under normal soil growth conditions. Hybrid lines can be created by crossing a line containing one or more PHB genes with a line containing the other gene(s) needed to complete the PHB biosynthetic pathway. Use of lines that possess cytoplasmic male sterility (Esser, K. et al., 2006, Progress in Botany, Springer Berlin Heidelberg. 67, 31-52) with the appropriate maintainer and restorer lines allows these hybrid lines to be produced efficiently. Cytoplasmic male sterility systems are already available for some Brassicaceae species (Esser, K. et al., 2006, Progress in Botany, Springer Berlin Heidelberg. 67, 31-52). These Brassicaceae species can be used as gene sources to produce cytoplasmic male sterility systems for other oilseeds of interest such as Camelina.

III. Methods for Use

The disclosed genetic constructs can be used to produce industrial oilseed plants for high levels of PHA production. Specifically, PHA is produced in the seed.

The transgenic plants can be grown and harvested. The polyhydroxyalkanoate can be isolated from the oilseeds and the remaining plant material can be used as a feedstock for industrial use, preferably for the production of oleochemicals, energy or for use as feed for animals. The polyhydroxyalkanoate harvested from the plants can then be used to produce plastics, rubber material, coating material, and binders for paints, or as a feedstock for producing chemical derivatives such as hydroxyacids, esters, alkenoic acids or amines. PHA also has several medical applications.

The present invention will be further understood by reference to the following non-limiting examples.

EXAMPLES Example 1 Design and Construction of Transformation Vectors for Production of PHB in Oilseeds

Five different vectors for seed specific expression of the PHB pathway were constructed containing different seed specific promoters for production of PHB in oilseeds (Table 1). Vector pMBXS490, a pCAMBIA based plasmid (Centre for Application of Molecular Biology to International Agriculture, Canberra, Australia), contains the following gene expression cassettes: (1) an expression cassette for PHA synthase containing the promoter from the soybean oleosin isoform A gene, a DNA fragment encoding the signal peptide of the small subunit of rubisco from pea (P. sativum) and the first 24 amino acids of the mature protein (Cashmore, A. R. 1983, In Genetic Engineering of Plants, pp. 29-38), a DNA fragment encoding a hybrid PHA synthase (PhaC; U.S. Pat. No. 6,316,262) in which the first nine amino acids at the N-terminus of this synthase are derived from the Pseudomonas oleovorans phaC1 gene and the remainder of the synthase coding sequence is derived from Zoogloea ramigera phaC gene, and the 3′ termination sequence from the soybean oleosin isoform A gene; (2) an expression cassette for reductase containing the promoter from the soybean oleosin isoform A gene, a DNA fragment encoding the signal peptide and the first 24 amino acids of the mature protein of the small subunit of rubisco from pea, a DNA fragment encoding a NADPH dependent reductase (PhaB) from Ralstonia eutropha eutropha (Peoples, O. & A. Sinskey, 1989, J. Biol. Chem., 264, 15293-15297), and the 3′ termination sequence from the soybean oleosin isoform A gene; (3) an expression cassette for thiolase containing the promoter from the soybean glycinin (gy1) gene (Iida et al., 1995, Plant Cell Reports, 14, 539-544), a DNA fragment encoding the signal peptide and the first 24 amino acids of the mature protein of the small subunit of rubisco from pea, the phaA gene encoding a β-ketothiolase (PhaA) from Ralstonia eutropha (Peoples, O. & A. Sinskey, 1989, J. Biol. Chem., 264, 15293-15297), and a 3′ termination sequence from the soybean glycinin gene; (4) an expression cassette for DsRed, a protein that can be visualized in seeds by placing them in light of the appropriate wavelength, containing the promoter from the cassava mosaic virus (CMV), a DNA fragment encoding a modified red fluorescent protein from Discosoma sp. (DsRed) in which eleven amino acids have been added to the C-terminus to increase solubility and/or prevent aggregation of the protein, and a termination sequence from the Agrobacterium tumefaciens nopaline synthase gene.

TABLE 1 Summary of transformation vectors containing seed specific promoters Promoter controlling Selectable or Plasmid expression of pha genes visible marker pMBXS490 Oleosin DsRed pMBXS364 LH DsRed pMBXS355 LH bar pMBXS491 Napin DsRed pMBXS492 Glycinin DsRed

Promoters are as follows: LH, promoter from the Lesquerella fendleri bifunctional oleate 12-hydroxylase:saturate gene (U.S. Pat. No. 6,437,220 B1); Oleosin, promoter from the soybean oleosin isoform A gene (Rowley and Herman, 1997, Biochim. Biophys. Acta 1345, 1-4); Napin, promoter from the Brassica napes napin gene (Ellenstrom, M. et al., 1996, Plant Molecular Biology, 32: 1019-1027); Glycinin, promoter from the soybean glycinin (gy1) gene (Iida, A. et al., 1995, Plant Cell Reports, 14:539-544).

Vectors pMBXS364, pMBXS355, pMBXS491, and pMBXS492 contain the same PHB pathway genes as pMBXS490 with the exception that the expression of these genes is under the control of different promoters as outlined in Table 1. Vector pMBXS355 contains an expression cassette for the bar gene, encoding phosphinothricin acetyltransferase whose expression is under the control of the 35S promoter. Expression of the bar gene allows selection of transformants based on their resistance to bialaphos. All other vectors in Table 1 contain expression cassettes for DsRed allowing the identification of transgenic seeds under the appropriate wavelength of light.

Example 2 Transformation of Camelina

In preparation for plant transformation experiments, seeds of Camelina sativa cultivar Suneson or Celine were sown directly into 4 inch pots filled with soil (Metro mix) in the greenhouse. Growth conditions were maintained at 24° C. during the day and 18° C. during the night. Plants were grown until flowering. Plants with a number of unopened flower buds were used in ‘floral dip’ transformations.

Agrobacterium strain GV3101 was transformed with the construct of interest using electroporation. A single colony of GV3101 containing the construct of interest was obtained from a freshly streaked plate and was inoculated into 5 mL LB medium. After overnight growth at 28° C., 2 mL of culture was transferred to a 500-mL flask containing 300 mL of LB and incubated overnight at 28° C. Cells were pelleted by centrifugation (6,000 rpm, 20 min), and diluted to an OD600 of ˜0.8 with infiltration medium containing 5% sucrose and 0.05% (v/v) Silwet-L77 (Lehle Seeds, Round Rock, Tex., USA). Camelina plants were transformed by “floral dip” using transformation constructs as follows. Pots containing plants at the flowering stage were placed inside a 460 mm height vacuum desiccator (Bel-Art, Pequannock, N.J., USA). Inflorescences were immersed into the Agrobacterium inoculum contained in a 500-ml beaker. A vacuum (85 kPa) was applied and held for 5 min. Plants were removed from the desiccator and were covered with plastic bags in the dark for 24 h at room temperature. Plants were removed from the bags and returned to normal growth conditions within the greenhouse for seed formation.

To identify Camelina seeds expressing DsRed, fully mature seeds were harvested from transformed plants and placed in a desiccator with anhydrous calcium sulfate as desiccant for at least 2 days prior to screening. DsRed expressing seeds were visualized in a darkroom with a green LumaMax LED flashlight (Lab Safety Supply, Inc., Janesville, Wis.) and a pair of KD's Dark Red glasses (Pacific Coast Sunglasses Inc., Santa Maria, Calif.).

To identify bialaphos resistant seeds, seeds from floral dip transformations were sterilized in 70% ethanol and 10% bleach, and washed in water. Sterilized seeds were placed on germination and selection medium in square Petri dishes. The germination and selection medium contained 10 mg/L bialaphos (Gold BioTechnology, 130178-500) in ½× MS medium, which was made with Murashige & Skoog medium mixture (Caisson Labs, MSP09) at half concentration. The plates were sealed and placed in a growth chamber for germination under a 16-h photoperiod, 3,000 lux light intensity, and temperatures of 23/20° C. at day/night. Seedlings with greenish cotyledons were picked and transferred to soil about six days after initiation of germination.

Example 3 Production of PHB in Seeds of Camelina

In initial transformation experiments with pMBXS490, 24 DsRed positive seeds were isolated. Four of these seeds were sacrificed to determine their PHB content using a previously described gas chromatography/butanolysis technique performed essentially as previously described (Somleva et al., 2008, Plant Biotechnol. J., 663-678). These four seeds contained 19.9, 12.0, 9.8, and 6.4% dwt PHB in the seed. When other seeds from this transformation were planted in soil, seedlings possessed whitish cotyledons and their growth was severely impaired. Only a few T₁ seeds with low levels of PHB were capable of germination and survival in soil in a greenhouse. These seedlings were still weak and possessed white or variegated cotyledons.

In transformations of pMBXS355 and pMBXS364, seeds from transformed plants were screened for resistance to bialophos and or visual screening for DsRed, respectively. Despite having the same promoter controlling the expression of the PHB biosynthetic pathway, the maximum PHB production in pMBXS355 (0.54% PHB) was significantly lower than the amount produced by pMBXS364 (3.4%) (Table 2). This is likely due to difficulty in distinguishing between weak pMBXS355 seedlings that produced higher levels of PHB and the non-transformed, bialophos sensitive seedlings.

TABLE 2 Comparison of PHB production in Lines isolated using bialaphos selection or visual screening Selectable or # of # of Lines w/ Range of PHB Screenable Lines PHB in T2 Production Vector Marker Tested Seeds (% seed weight) pMBXS355 Bar¹ 204 5 0.05 to 0.54% pMBXS364 DsRed² 170 85 0.5 to 3.4% ¹Selection of transformants performed by germination of seeds on tissue culture plates containing 10 mg/L bialophos. ²Selection of transformants performed by visual screening for DsRed expression.

In transformations with pMBX491 and pMBX492 containing the PHB genes under the control of the napin and glycinin promoters, respectively, were healthier than transformants obtained from pMBX490 transformations. For pMBX491, T2 seeds were isolated containing 8% PHB in DsRed seeds picked from the segregating population. These seeds possessed a 75% germination rate and a 60% survival rate under greenhouse conditions in soil. The cotyledons after 11 days were chlorotic and the growth of this line was significantly delayed compared to wild-type. For pMBX492, T2 seeds were isolated containing 6.9% PHB in DsRed seeds picked from the segregating population. These seeds possessed a 75% germination rate and a 70% survival rate under greenhouse conditions in soil. After 11 days, the cotyledons and first true leaves of this transformant were green. The growth of this line was somewhat delayed compared to wild-type but faster than the pMBXS491 line.

The 19% dwt PHB produced in a single seed obtained from Camelina plants transformed with construct pMBXS490 was an unexpected result and is the highest level of PHB reported in oilseeds to date. Previous studies with Brassica napus produced up to 7.7% dwt PHB. These seeds were obtained from transformation of Brassica napus using stem segments as the explants and selection of the transformed explants (Fry, J. et al., 1987, 6, 321-325) using glyphosate resistance obtained from expression of a gene encoding 5-enolpyruvylshikimate-3-phosphate synthase. Researchers did not report any germination issues with seeds isolated from the transformed plants [Houmiel et al., 1999, Planta, 209, 547-550; Valentin et al., 1999, Int. J. Biol. Macromol. 25, 303-306].

The use of DsRed as a visual marker in Camelina enabled the identification of high PHB producing seeds that would not have germinated in a typical seed screening procedure where an antibiotic or herbicide selectable marker, such as glyphosate resistance, is employed to provide resistance to the selection agent during seed germination and seedling development in tissue culture medium.

Example 4 Transformation of Brassica napus, Brassica carinata, and Brassica juncea

Transformation of Brassica carinata

Brassica carinata can be transformed using a previously described floral dip method (Shiv et al., 2008, Journal of Plant Biochemistry and Biotechnology 17, 1-4). Briefly constructs of interest are transformed into Agrobacterium strain GV-3101 and cells are grown in liquid medium. Cells are harvested and resuspended in a transformation medium consisting of ½ MS salts, 5% sucrose, and 0.05% Silwet L-77. Brassica carinata plants are grown in a greenhouse until inflorescences develop and approximately 25% of their flowers are opened. Plants are submerged in the prepared Agrobacterium solution for approximately 1 minute, and covered for 24 hours. Plants are returned to the greenhouse and allowed to set seed. Transformed seeds are screened by picking DsRed seeds under the appropriate wavelength of light as described above.

Transformation of Brassica napus

Brassica seeds are surface sterilized in 10% commercial bleach (Javex, Colgate-Palmolive) for 30 min with gentle shaking. The seeds are washed three times in sterile distilled water and placed in germination medium comprising Murashige-Skoog (MS) salts and vitamins, 3% (w/v) sucrose and 0.7% (w/v) phytagar, pH 5.8 at a density of 20 per plate and maintained at 24° C. an a 16 h light/8 h dark photoperiod at a light intensity of 60-80 μEm⁻² s⁻¹ for 4-5 days.

Constructs of interest are introduced into Agrobacterium tumefacians strain EHA101 (Hood et. al., 1986, J. Bacteriol. 168: 1291-1301) by electroporation. Prior to transformation of cotyledonary petioles, single colonies of strain EHA101 harboring each construct are grown in 5 ml of minimal medium supplemented with appropriate antibiotics for 48 hr at 28° C. One ml of bacterial suspension was pelleted by centrifugation for 1 min in a microfuge. The pellet was resuspended in 1 ml minimal medium.

For transformation, cotyledons are excised from 4 or in some cases 5 day old seedlings so that they included ˜2 mm of petiole at the base. Individual cotyledons with the cut surface of their petioles are immersed in diluted bacterial suspension for 1 s and immediately embedded to a depth of ˜2 mm in co-cultivation medium, MS medium with 3% (w/v) sucrose and 0.7% phytagar and enriched with 20 μM benzyladenine. The inoculated cotyledons are plated at a density of 10 per plate and incubated under the same growth conditions for 48 h. After co-cultivation, the cotyledons are transferred to regeneration medium comprising MS medium supplemented with 3% sucrose, 20 μM benzyladenine, 0.7% (w/v) phytagar, pH 5.8, 300 mg/L timentinin and 20 mg/L kanamycin sulfate.

After 2-3 weeks regenerant shoots obtained are cut and maintained on “shoot elongation” medium (MS medium containing, 3% sucrose, 300 mg/L timentin, 0.7% (w/v) phytagar, 300 mg/L timentinin and 20 mg/L kanamycin sulfate, pH 5.8) in Magenta jars. The elongated shoots are transferred to “rooting” medium comprising MS medium, 3% sucrose, 2 mg/L indole butyric acid, 0.7% phytagar and 500 mg/L carbenicillin. After roots emerge, plantlets are transferred to potting mix (Redi Earth, W.R. Grace and Co.). The plants are maintained in a misting chamber (75% relative humidity) under the same growth conditions. Plants are allowed to self pollinate to produce seeds. Seeds are screened by visualization of DsRed as described above.

Brassica napus can also be transformed using the floral dip procedure described by Shiv et al. (Shiv et al., 2008, Journal of Plant Biochemistry and Biotechnology 17, 1-4) as described above for Brassica carinata.

Transformation of Brassica juncea

Brassica juncea can be transformed using hypocotyl explants according to the methods described by Barfield and Pua (Barfield and Pua, Plant Cell Reports, 10, 308-314) or Pandian et al. (Pandian, et al., 2006, Plant Molecular Biology Reporter 24: 103a-103i) as follows.

B. juncea seeds are sterilized 2 min in 70% (v/v) ethanol and washed for 20 min in 25% commercial bleach (10 g/L hypochlorite). Seeds are rinsed 3× in sterile water. Surface-sterilized seeds are plated on germination medium (1× MS salts, 1×MS vitamins, 30 g/L sucrose, 500 mg/L MES. pH 5.5) and kept in the cold room for 2 days. Seeds are incubated for 4-6 days at 24° C. under low light (20 μm m⁻¹s⁻¹). Hypocotyl segments are excised and rinsed in 50 mL of callus induction medium (1× MS salts, 1× B5 vitamins, 30 g/L sucrose, 500 mg/L MES, 1.0 mg/L 2.4-D, 1.0 mg/L kinetin pH 5.8) for 30 min without agitation. This procedure is repeated but with agitation on orbital shaker (˜140 g) for 48 h at 24° C. in low light (10 μm m⁻¹s⁻¹).

Agrobacterium can be prepared as follows: Cells of Agrobacterium strain AGL1 (Lazo, G. et al. (1991), Biotechnology, 9: 963-967) containing the construct of interest are grown in 5 mL of LB medium with appropriate antibiotic at 28° C. for 2 days. The 5 mL culture is transferred to 250 mL flask with 45 mL of LB and cultured for 4 h at 28° C. Cells is pelleted and resuspended in BM medium (1× MS salts, 1× B5 vitamins, 30 g/L sucrose, 500 mg/L MES, pH 5.8). The optical density at 600 nm is adjusted to 0.2 with BM medium and used for inoculation.

Explants are cocultivated with Agrobacterium for 20 min after which time the Agrobacterium suspension is removed. Hypocotyl explants are washed once in callus induction medium after which cocultivation proceeds for 48 h with gentle shaking on orbital shaker. After several washes in CIM, explants are transferred to selective shoot-inducing medium (500 mg/L AgNO2, 0.4 mg/L zeatin riboside, 2.0 mg/L benzylamino purine, 0.01 mg/L GA, 200 mg/L Timentin appropriate selection agent and 8 g/L agar added to basal medium) plates for regeneration at 24° C. Root formation is induced on root-inducing medium (0.5×MS salts, 0.5× B5 vitamins, 10 g/L sucrose, 500 mg/L MES, 0.1 mg/L indole-3-butyric acid, 200 mg/L Timentin, appropriate selection agent and S g/L agar, pH 5.8).

Plantlets are transferred to are removed from agar, gently washed, and transferred to potting soil in pots. Plants are grown in a humid environment for a week and then transferred to the greenhouse.

Example 5 Production of Hybrid Lines that are not Capable of Germinating

In previous experiments in Arabidopsis, lower levels of PHB were obtained when lines expressing individual PHB genes were crossed to produce a plant containing the entire PHB biosynthetic pathway (Nawrath, C., Y. Poirier, et al., 1994, Proc. Natl. Acad. Sci. USA 91, 12760-12764) than when multi-gene constructs containing the entire PHB biosynthetic pathway were constructed and transformed (Bohmert, K., I. et al., 2000, Planta 211, 841-845;U.S. Pat. No. 6,448,473). This observation led to the subsequent predominant use of multi-gene constructs for PHB production in plants. However, in some scenarios, it may be advantageous to insert a multi-gene pathway into the plant by crossing of lines containing portions of the pathway to produce hybrid plants in which the entire pathway has been reconstructed. This is especially the case when high levels of product in a seed compromises the ability of the seed to germinate or the resulting seedling to survive under normal soil growth conditions. Hybrid lines can be created by crossing a line containing one or more PHB genes with a line containing the other gene(s) needed to complete the PHB biosynthetic pathway. Use of lines that possess cytoplasmic male sterility (Esser, K. et al., 2006, Progress in Botany, Springer Berlin Heidelberg. 67, 31-52) with the appropriate maintainer and restorer lines allows these hybrid lines to be produced efficiently. Cytoplasmic male sterility systems are already available for some Brassicaceae species (Esser, K. et al., 2006, Progress in Botany, Springer Berlin Heidelberg. 67, 31-52). These Brassicaceae species can be used as gene sources to produce cytoplasmic male sterility systems for other oilseeds of interest such as Camelina. Cytoplasmic male sterility has also been reported upon expression of a β-ketothiolase from the chloroplast genome in tobacco (Ruiz, O. N. and H. Daniell, 2005, Plant Physiol. 138, 1232-1246). Male sterility has also been reported upon expression of the faoA gene encoding the α-subunit of the fatty acid β-oxidation complex from Pseudomonas putida (U.S. Pat. No. 6,586,658).

High PHB producing lines that are not capable of germination can be produced using oilseed lines that possess cytoplasmic male sterility (CMS) controlled by an extranuclear genome (i.e. mitochondria or chloroplast). The male sterile line is typically maintained by crossing with a maintainer line that is genetically identical except that it possesses normal fertile cytoplasm and is therefore male fertile. Transformation of the maintainer line with one or more genes for the PHB biosynthetic pathway and crossing this modified maintainer line with the original male sterile line will produce a male sterile line possessing a portion of the PHB biosynthetic pathway. In this example, insertion of the phaA and phaC genes into the maintainer line and crossing with the original male cytoplasmic sterile line will form a male sterile line containing the phaA and phaC genes.

Fertility can be restored to this line using a “restorer line” that carries the appropriate nuclear restorer genes. Alternatively, the restorer line can be transformed with the remaining genes required to complete the PHB biosynthetic pathway and crossed with the previously created male sterile line containing phaA and phaC to produce a hybrid line containing the entire PHB biosynthetic pathway.

Crosses can be performed in the field by planting multiple rows of the male sterile line, the line that will produce the seed, next to a few rows of the male fertile line. Harvested seed can be used for subsequent plantings or as the PHB containing seed for crushing and extraction. When expression cassettes for the PHB genes in this example are controlled by strong promoters, such as the soybean oleosin promoter, high PHB producing seeds generated in this manner will possess weak seedlings upon germination and will not be able to survive field conditions under normal growth circumstances unless treated with a material that promotes seedling strength/vigor. This adds a level of gene containment.

Cytoplasmic male sterility systems are already available for some Brassicaceae species (Esser, K., 2006, Progress in Botany, Springer Berlin Heidelberg. 67, 31-52). These Brassicaceae species can be used as gene sources to produce cytoplasmic male sterility systems for other oilseeds of interest such as Camelina. Cytoplasmic male sterility has also been reported upon expression of a β-ketothiolase from the chloroplast genome in tobacco (Ruiz, O. N. and H. Daniell, 2005, Plant Physiol. 138, 1232-1246). Overexpression of β-ketothiolase in Camelina to generate a male sterile line and subsequent crossing with a line expressing phaB and phaC could also be used for hybrid seed production.

Male sterile lines have also been produced in Brassica napus by overexpression of the faoA gene from Pseudomonas putida under the control of the phaseolin promoter sequence (U.S. Pat. No. 6,586,658).

Double haploid technology can be used to speed up the breeding process. In the double haploid technique, immature pollen grains (haploids) are exposed to treatments that result in doubling of the existing genetic material resulting in homozygous, true breeding material in a single generation.

The references, patents, and patent applications cited throughout are incorporated by reference where permissible in their entireties.

Vector: pMBXS490 (SEQ ID NO: 1) 1 GGGGATCCGT ACGTAAGTAC GTACTCAAAA TGCCAACAAA TAAAAAAAAA 51 GTTGCTTTAA TAATGCCAAA ACAAATTAAT AAAACACTTA CAACACCGGA 101 TTTTTTTTAA TTAAAATGTG CCATTTAGGA TAAATAGTTA ATATTTTTAA 151 TAATTATTTA AAAAGCCGTA TCTACTAAAA TGATTTTTAT TTGGTTGAAA 201 ATATTAATAT GTTTAAATCA ACACAATCTA TCAAAATTAA ACTAAAAAAA 251 AAATAAGTGT ACGTGGTTAA CATTAGTACA GTAATATAAG AGGAAAATGA 301 GAAATTAAGA AATTGAAAGC GAGTCTAATT TTTAAATTAT GAACCTGCAT 351 ATATAAAAGG AAAGAAAGAA TCCAGGAAGA AAAGAAATGA AACCATGCAT 401 GGTCCCCTCG TCATCACGAG TTTCTGCCAT TTGCAATAGA AACACTGAAA 451 CACCTTTCTC TTTGTCACTT AATTGAGATG CCGAAGCCAC CTCACACCAT 501 GAACTTCATG AGGTGTAGCA CCCAAGGCTT CCATAGCCAT GCATACTGAA 551 GAATGTCTCA AGCTCAGCAC CCTACTTCTG TGACGTGTCC CTCATTCACC 601 TTCCTCTCTT CCCTATAAAT AACCACGCCT CAGGTTCTCC GCTTCACAAC 651 TCAAACATTC TCTCCATTGG TCCTTAAACA CTCATCAGTC ATCACCGCGG 701 CCGCGGAATT CATGGCTTCT ATGATATCCT CTTCCGCTGT GACAACAGTC 751 AGCCGTGCCT CTAGGGGGCA ATCCGCCGCA GTGGCTCCAT TCGGCGGCCT 801 CAAATCCATG ACTGGATTCC CAGTGAAGAA GGTCAACACT GACATTACTT 851 CCATTACAAG CAATGGTGGA AGAGTAAAGT GCATGCAGGT GTGGCCTCCA 901 ATTGGAAAGA AGAAGTTTGA GACTCTTTCC TATTTGCCAC CATTGACGAG 951 AGATTCTAGA GTGACTGACG TTGTCATCGT ATCCGCCGCC CGCACCGCGG 1001 TCGGCAAGTT TGGCGGCTCG CTGGCCAAGA TCCCGGCACC GGAACTGGGT 1051 GCCGTGGTCA TCAAGGCCGC GCTGGAGCGC GCCGGCGTCA AGCCGGAGCA 1101 GGTGAGCGAA GTCATCATGG GCCAGGTGCT GACCGCCGGT TCGGGCCAGA 1151 ACCCCGCACG CCAGGCCGCG ATCAAGGCCG GCCTGCCGGC GATGGTGCCG 1201 GCCATGACCA TCAACAAGGT GTGCGGCTCG GGCCTGAAGG CCGTGATGCT 1251 GGCCGCCAAC GCGATCATGG CGGGCGACGC CGAGATCGTG GTGGCCGGCG 1301 GCCAGGAAAA CATGAGCGCC GCCCCGCACG TGCTGCCGGG CTCGCGCGAT 1351 GGTTTCCGCA TGGGCGATGC CAAGCTGGTC GACACCATGA TCGTCGACGG 1401 CCTGTGGGAC GTGTACAACC AGTACCACAT GGGCATCACC GCCGAGAACG 1451 TGGCCAAGGA ATACGGCATC ACACGCGAGG CGCAGGATGA GTTCGCCGTC 1501 GGCTCGCAGA ACAAGGCCGA AGCCGCGCAG AAGGCCGGCA AGTTTGACGA 1551 AGAGATCGTC CCGGTGCTGA TCCCGCAGCG CAAGGGCGAC CCGGTGGCCT 1601 TCAAGACCGA CGAGTTCGTG CGCCAGGGCG CCACGCTGGA CAGCATGTCC 1651 GGCCTCAAGC CCGCCTTCGA CAAGGCCGGC ACGGTGACCG CGGCCAACGC 1701 CTCGGGCCTG AACGACGGCG CCGCCGCGGT GGTGGTGATG TCGGCGGCCA 1751 AGGCCAAGGA ACTGGGCCTG ACCCCGCTGG CCACGATCAA GAGCTATGCC 1801 AACGCCGGTG TCGATCCCAA GGTGATGGGC ATGGGCCCGG TGCCGGCCTC 1851 CAAGCGCGCC CTGTCGCGCG CCGAGTGGAC CCCGCAAGAC CTGGACCTGA 1901 TGGAGATCAA CGAGGCCTTT GCCGCGCAGG CGCTGGCGGT GCACCAGCAG 1951 ATGGGCTGGG ACACCTCCAA GGTCAATGTG AACGGCGGCG CCATCGCCAT 2001 CGGCCACCCG ATCGGCGCGT CGGGCTGCCG TATCCTGGTG ACGCTGCTGC 2051 ACGAGATGAA GCGCCGTGAC GCGAAGAAGG GCCTGGCCTC GCTGTGCATC 2101 GGCGGCGGCA TGGGCGTGGC GCTGGCAGTC GAGCGCAAAT AACTCGAGGC 2151 GGCCGCAGCC CTTTTTGTAT GTGCTACCCC ACTTTTGTCT TTTTGGCAAT 2201 AGTGCTAGCA ACCAATAAAT AATAATAATA ATAATGAATA AGAAAACAAA 2251 GGCTTTAGCT TGCCTTTTGT TCACTGTAAA ATAATAATGT AAGTACTCTC 2301 TATAATGAGT CACGAAACTT TTGCGGGAAT AAAAGGAGAA ATTCCAATGA 2351 GTTTTCTGTC AAATCTTCTT TTGTCTCTCT CTCTCTCTCT TTTTTTTTTT 2401 TCTTTCTTCT GAGCTTCTTG CAAAACAAAA GGCAAACAAT AACGATTGGT 2451 CCAATGATAG TTAGCTTGAT CGATGATATC TTTAGGAAGT GTTGGCAGGA 2501 CAGGACATGA TGTAGAAGAC TAAAATTGAA AGTATTGCAG ACCCAATAGT 2551 TGAAGATTAA CTTTAAGAAT GAAGACGTCT TATCAGGTTC TTCATGACTT 2601 AAGCTTTAAG AGGAGTCCAC CATGGTAGAT CTGACTAGTA GAAGGTAATT 2651 ATCCAAGATG TAGCATCAAG AATCCAATGT TTACGGGAAA AACTATGGAA 2701 GTATTATGTG AGCTCAGCAA GAAGCAGATC AATATGCGGC ACATATGCAA 2751 CCTATGTTCA AAAATGAAGA ATGTACAGAT ACAAGATCCT ATACTGCCAG 2801 AATACGAAGA AGAATACGTA GAAATTAAGA AAGAAGAACC AGGCGAAGAA 2851 AAGAATCTTG AAGACGTAAG CACTGACGAC AACACTGAAA AGAAGAAGAT 2901 AAGGTCGGTG ATTGTGAAAG AGACATAGAG GACACATGTA AGGTGGAAAA 2951 TGTAAGGGCG GAAAGTAACC TTATCACAAA GGAATCTTAT CCCCCACTAC 3001 TTATCCTTTT ATATTTTTCC GTGTCATTTT TGCCCTTGAG TTTTCCTATA 3051 TAAGGAACCA AGTTCGGCAT TTGTGAAAAC AAGAAAAAAT TGGTGTAAGC 3101 TATTTTCTTT GAAGTACTGA GGATACAACT TCAGAGAAAT TTGTAAGAAA 3151 GTGGATCGAA ACCATGGCCT CCTCCGAGAA CGTCATCACC GAGTTCATGC 3201 GCTTCAAGGT GCGCATGGAG GGCACCGTGA ACGGCCACGA GTTCGAGATC 3251 GAGGGCGAGG GCGAGGGCCG CCCCTACGAG GGCCACAACA CCGTGAAGCT 3301 GAAGGTGACC AAGGGCGGCC CCCTGCCCTT CGCCTGGGAC ATCCTGTCCC 3351 CCCAGTTCCA GTACGGCTCC AAGGTGTACG TGAAGCACCC CGCCGACATC 3401 CCCGACTACA AGAAGCTGTC CTTCCCCGAG GGCTTCAAGT GGGAGCGCGT 3451 GATGAACTTC GAGGACGGCG GCGTGGCGAC CGTGACCCAG GACTCCTCCC 3501 TGCAGGACGG CTGCTTCATC TACAAGGTGA AGTTCATCGG CGTGAACTTC 3551 CCCTCCGACG GCCCCGTGAT GCAGAAGAAG ACCATGGGCT GGGAGGCCTC 3601 CACCGAGCGC CTGTACCCCC GCGACGGCGT GCTGAAGGGC GAGACCCACA 3651 AGGCCCTGAA GCTGAAGGAC GGCGGCCACT ACCTGGTGGA GTTCAAGTCC 3701 ATCTACATGG CCAAGAAGCC CGTGCAGCTG CCCGGCTACT ACTACGTGGA 3751 CGCCAAGCTG GACATCACCT CCCACAACGA GGACTACACC ATCGTGGAGC 3801 AGTACGAGCG CACCGAGGGC CGCCACCACC TGTTCCTGGT ACCAATGAGC 3851 TCTGTCCAAC AGTCTCAGGG TTAATGTCTA TGTATCTTAA ATAATGTTGT 3901 CGGCGATCGT TCAAACATTT GGCAATAAAG TTTCTTAAGA TTGAATCCTG 3951 TTGCCGGTCT TGCGATGATT ATCATATAAT TTCTGTTGAA TTACGTTAAG 4001 CATGTAATAA TTAACATGTA ATGCATGACG TTATTTATGA GATGGGTTTT 4051 TATGATTAGA GTCCCGCAAT TATACATTTA ATACGCGATA GAAAACAAAA 4101 TATAGCGCGC AAACTAGGAT AAATTATCGC GCGCGGTGTC ATCTATGTTA 4151 CTAGATCGGG AATTAAACTA TCAGTGTTTG ACAGGATATA TTGGCGGGTA 4201 AACCTAAGAG AAAAGAGCGT TTATTAGAAT AACGGATATT TAAAAGGGCG 4251 TGAAAAGGTT TATCCGTTCG TCCATTTGTA TGTGCATGCC AACCACAGGG 4301 TTCCCCTCGG GATCAAAGTA CTTTGATCCA ACCCCTCCGC TGCTATAGTG 4351 CAGTCGGCTT CTGACGTTCA GTGCAGCCGT CTTCTGAAAA CGACATGTCG 4401 CACAAGTCCT AAGTTACGCG ACAGGCTGCC GCCCTGCCCT TTTCCTGGCG 4451 TTTTCTTGTC GCGTGTTTTA GTCGCATAAA GTAGAATACT TGCGACTAGA 4501 ACCGGAGACA TTACGCCATG AACAAGAGCG CCGCCGCTGG CCTGCTGGGC 4551 TATGCCCGCG TCAGCACCGA CGACCAGGAC TTGACCAACC AACGGGCCGA 4601 ACTGCACGCG GCCGGCTGCA CCAAGCTGTT TTCCGAGAAG ATCACCGGCA 4651 CCAGGCGCGA CCGCCCGGAG CTGGCCAGGA TGCTTGACCA CCTACGCCCT 4701 GGCGACGTTG TGACAGTGAC CAGGCTAGAC CGCCTGGCCC GCAGCACCCG 4751 CGACCTACTG GACATTGCCG AGCGCATCCA GGAGGCCGGC GCGGGCCTGC 4801 GTAGCCTGGC AGAGCCGTGG GCCGACACCA CCACGCCGGC CGGCCGCATG 4851 GTGTTGACCG TGTTCGCCGG CATTGCCGAG TTCGAGCGTT CCCTAATCAT 4901 CGACCGCACC CGGAGCGGGC GCGAGGCCGC CAAGGCCCGA GGCGTGAAGT 4951 TTGGCCCCCG CCCTACCCTC ACCCCGGCAC AGATCGCGCA CGCCCGCGAG 5001 CTGATCGACC AGGAAGGCCG CACCGTGAAA GAGGCGGCTG CACTGCTTGG 5051 CGTGCATCGC TCGACCCTGT ACCGCGCACT TGAGCGCAGC GAGGAAGTGA 5101 CGCCCACCGA GGCCAGGCGG CGCGGTGCCT TCCGTGAGGA CGCATTGACC 5151 GAGGCCGACG CCCTGGCGGC CGCCGAGAAT GAACGCCAAG AGGAACAAGC 5201 ATGAAACCGC ACCAGGACGG CCAGGACGAA CCGTTTTTCA TTACCGAAGA 5251 GATCGAGGCG GAGATGATCG CGGCCGGGTA CGTGTTCGAG CCGCCCGCGC 5301 ACGTCTCAAC CGTGCGGCTG CATGAAATCC TGGCCGGTTT GTCTGATGCC 5351 AAGCTGGCGG CCTGGCCGGC CAGCTTGGCC GCTGAAGAAA CCGAGCGCCG 5401 CCGTCTAAAA AGGTGATGTG TATTTGAGTA AAACAGCTTG CGTCATGCGG 5451 TCGCTGCGTA TATGATGCGA TGAGTAAATA AACAAATACG CAAGGGGAAC 5501 GCATGAAGGT TATCGCTGTA CTTAACCAGA AAGGCGGGTC AGGCAAGACG 5551 ACCATCGCAA CCCATCTAGC CCGCGCCCTG CAACTCGCCG GGGCCGATGT 5601 TCTGTTAGTC GATTCCGATC CCCAGGGCAG TGCCCGCGAT TGGGCGGCCG 5651 TGCGGGAAGA TCAACCGCTA ACCGTTGTCG GCATCGACCG CCCGACGATT 5701 GACCGCGACG TGAAGGCCAT CGGCCGGCGC GACTTCGTAG TGATCGACGG 5751 AGCGCCCCAG GCGGCGGACT TGGCTGTGTC CGCGATCAAG GCAGCCGACT 5801 TCGTGCTGAT TCCGGTGCAG CCAAGCCCTT ACGACATATG GGCCACCGCC 5851 GACCTGGTGG AGCTGGTTAA GCAGCGCATT GAGGTCACGG ATGGAAGGCT 5901 ACAAGCGGCC TTTGTCGTGT CGCGGGCGAT CAAAGGCACG CGCATCGGCG 5951 GTGAGGTTGC CGAGGCGCTG GCCGGGTACG AGCTGCCCAT TCTTGAGTCC 6001 CGTATCACGC AGCGCGTGAG CTACCCAGGC ACTGCCGCCG CCGGCACAAC 6051 CGTTCTTGAA TCAGAACCCG AGGGCGACGC TGCCCGCGAG GTCCAGGCGC 6101 TGGCCGCTGA AATTAAATCA AAACTCATTT GAGTTAATGA GGTAAAGAGA 6151 AAATGAGCAA AAGCACAAAC ACGCTAAGTG CCGGCCGTCC GAGCGCACGC 6201 AGCAGCAAGG CTGCAACGTT GGCCAGCCTG GCAGACACGC CAGCCATGAA 6251 GCGGGTCAAC TTTCAGTTGC CGGCGGAGGA TCACACCAAG CTGAAGATGT 6301 ACGCGGTACG CCAAGGCAAG ACCATTACCG AGCTGCTATC TGAATACATC 6351 GCGCAGCTAC CAGAGTAAAT GAGCAAATGA ATAAATGAGT AGATGAATTT 6401 TAGCGGCTAA AGGAGGCGGC ATGGAAAATC AAGAACAACC AGGCACCGAC 6451 GCCGTGGAAT GCCCCATGTG TGGAGGAACG GGCGGTTGGC CAGGCGTAAG 6501 CGGCTGGGTT GTCTGCCGGC CCTGCAATGG CACTGGAACC CCCAAGCCCG 6551 AGGAATCGGC GTGACGGTCG CAAACCATCC GGCCCGGTAC AAATCGGCGC 6601 GGCGCTGGGT GATGACCTGG TGGAGAAGTT GAAGGCCGCG CAGGCCGCCC 6651 AGCGGCAACG CATCGAGGCA GAAGCACGCC CCGGTGAATC GTGGCAAGCG 6701 GCCGCTGATC GAATCCGCAA AGAATCCCGG CAACCGCCGG CAGCCGGTGC 6751 GCCGTCGATT AGGAAGCCGC CCAAGGGCGA CGAGCAACCA GATTTTTTCG 6801 TTCCGATGCT CTATGACGTG GGCACCCGCG ATAGTCGCAG CATCATGGAC 6851 GTGGCCGTTT TCCGTCTGTC GAAGCGTGAC CGACGAGCTG GCGAGGTGAT 6901 CCGCTACGAG CTTCCAGACG GGCACGTAGA GGTTTCCGCA GGGCCGGCCG 6951 GCATGGCCAG TGTGTGGGAT TACGACCTGG TACTGATGGC GGTTTCCCAT 7001 CTAACCGAAT CCATGAACCG ATACCGGGAA GGGAAGGGAG ACAAGCCCGG 7051 CCGCGTGTTC CGTCCACACG TTGCGGACGT ACTCAAGTTC TGCCGGCGAG 7101 CCGATGGCGG AAAGCAGAAA GACGACCTGG TAGAAACCTG CATTCGGTTA 7151 AACACCACGC ACGTTGCCAT GCAGCGTACG AAGAAGGCCA AGAACGGCCG 7201 CCTGGTGACG GTATCCGAGG GTGAAGCCTT GATTAGCCGC TACAAGATCG 7251 TAAAGAGCGA AACCGGGCGG CCGGAGTACA TCGAGATCGA GCTAGCTGAT 7301 TGGATGTACC GCGAGATCAC AGAAGGCAAG AACCCGGACG TGCTGACGGT 7351 TCACCCCGAT TACTTTTTGA TCGATCCCGG CATCGGCCGT TTTCTCTACC 7401 GCCTGGCACG CCGCGCCGCA GGCAAGGCAG AAGCCAGATG GTTGTTCAAG 7451 ACGATCTACG AACGCAGTGG CAGCGCCGGA GAGTTCAAGA AGTTCTGTTT 7501 CACCGTGCGC AAGCTGATCG GGTCAAATGA CCTGCCGGAG TACGATTTGA 7551 AGGAGGAGGC GGGGCAGGCT GGCCCGATCC TAGTCATGCG CTACCGCAAC 7601 CTGATCGAGG GCGAAGCATC CGCCGGTTCC TAATGTACGG AGCAGATGCT 7651 AGGGCAAATT GCCCTAGCAG GGGAAAAAGG TCGAAAAGGT CTCTTTCCTG 7701 TGGATGTACC GTACATTGGG AACCCAAAGC CGTACATTGG GAACCGGAAC 7751 CCGTACATTG GGAACCCAAA GCCGTACATT GGGAACCGGT CACACATGTA 7801 AGTGACTGAT ATAAAAGAGA AAGAAGGCCA TTTTTCCGCC TAAAACTCTT 7851 TAAAACTTAT TAAAACTCTT AAAACCCGCC TGGCCTGTGC ATAACTGTCT 7901 GGCCAGCGCA CAGCCGAAGA GCTGCAAAAA GCGCCTACCC TTCGGTCGCT 7951 GCGCTCCCTA CGCCCCGCCG CTTCGCGTCG GCCTATCGCG GCCGCTGGCC 8001 GCTCAAAAAT GGCTGGCCTA CGGCCAGGCA ATCTACCAGG GCGCGGACAA 8051 GCCGCGCCGT CGCCACTCGA CCGCCGGCGC CCACATCAAG GCACCCTGCC 8101 TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG 8151 GAGACGGTCA CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG 8201 TCAGGGCGCG TCAGCGGGTG TTGGCGGGTG TCGGGGCGCA GCCATGACCC 8251 AGTCACGTAG CGATAGCGGA GTGTATACTG GCTTAACTAT GCGGCATCAG 8301 AGCAGATTGT ACTGAGAGTG CACCATATGC GGTGTGAAAT ACCGCACAGA 8351 TGCGTAAGGA GAAAATACCG CATCAGGCGC TCTTCCGCTT CCTCGCTCAC 8401 TGACTCGCTG CGCTCGGTCG TTCGGCTGCG GCGAGCGGTA TCAGCTCACT 8451 CAAAGGCGGT AATACGGTTA TCCACAGAAT CAGGGGATAA CGCAGGAAAG 8501 AACATGTGAG CAAAAGGCCA GCAAAAGGCC AGGAACCGTA AAAAGGCCGC 8551 GTTGCTGGCG TTTTTCCATA GGCTCCGCCC CCCTGACGAG CATCACAAAA 8601 ATCGACGCTC AAGTCAGAGG TGGCGAAACC CGACAGGACT ATAAAGATAC 8651 CAGGCGTTTC CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG TTCCGACCCT 8701 GCCGCTTACC GGATACCTGT CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC 8751 TTTCTCATAG CTCACGCTGT AGGTATCTCA GTTCGGTGTA GGTCGTTCGC 8801 TCCAAGCTGG GCTGTGTGCA CGAACCCCCC GTTCAGCCCG ACCGCTGCGC 8851 CTTATCCGGT AACTATCGTC TTGAGTCCAA CCCGGTAAGA CACGACTTAT 8901 CGCCACTGGC AGCAGCCACT GGTAACAGGA TTAGCAGAGC GAGGTATGTA 8951 GGCTGGCCTA CAGAGTTCTT GAAGTGGTGG CCTAACTACG GCTACACTAG 9001 AAGGACAGTA TTTGGTATCT GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA 9051 AAAGAGTTGG TAGCTCTTGA TCCGGCAAAC AAACCACCGC TGGTAGCGGT 9101 GGTTTTTTTG TTTGCAAGCA GCAGATTACG CGCAGAAAAA AAGGATCTCA 9151 AGAAGATCCT TTGATCTTTT CTACGGGGTC TGACGCTCAG TGGAACGAAA 9201 ACTCACGTTA AGGGATTTTG GTCATGCATT CTAGGTACTA AAACAATTCA 9251 TCCAGTAAAA TATAATATTT TATTTTCTCC CAATCAGGCT TGATCCCCAG 9301 TAAGTCAAAA AATAGCTCGA CATACTGTTC TTCCCCGATA TCCTCCCTGA 9351 TCGACCGGAC GCAGAAGGCA ATGTCATACC ACTTGTCCGC CCTGCCGCTT 9401 CTCCCAAGAT CAATAAAGCC ACTTACTTTG CCATCTTTCA CAAAGATGTT 9451 GCTGTCTCCC AGGTCGCCGT GGGAAAAGAC AAGTTCCTCT TCGGGCTTTT 9501 CCGTCTTTAA AAAATCATAC AGCTCGCGCG GATCTTTAAA TGGAGTGTCT 9551 TCTTCCCAGT TTTCGCAATC CACATCGGCC AGATCGTTAT TCAGTAAGTA 9601 ATCCAATTCG GCTAAGCGGC TGTCTAAGCT ATTCGTATAG GGACAATCCG 9651 ATATGTCGAT GGAGTGAAAG AGCCTGATGC ACTCCGCATA CAGCTCGATA 9701 ATCTTTTCAG GGCTTTGTTC ATCTTCATAC TCTTCCGAGC AAAGGACGCC 9751 ATCGGCCTCA CTCATGAGCA GATTGCTCCA GCCATCATGC CGTTCAAAGT 9801 GCAGGACCTT TGGAACAGGC AGCTTTCCTT CCAGCCATAG CATCATGTCC 9851 TTTTCCCGTT CCACATCATA GGTGGTCCCT TTATACCGGC TGTCCGTCAT 9901 TTTTAAATAT AGGTTTTCAT TTTCTCCCAC CAGCTTATAT ACCTTAGCAG 9951 GAGACATTCC TTCCGTATCT TTTACGCAGC GGTATTTTTC GATCAGTTTT 10001 TTCAATTCCG GTGATATTCT CATTTTAGCC ATTTATTATT TCCTTCCTCT 10051 TTTCTACAGT ATTTAAAGAT ACCCCAAGAA GCTAATTATA ACAAGACGAA 10101 CTCCAATTCA CTGTTCCTTG CATTCTAAAA CCTTAAATAC CAGAAAACAG 10151 CTTTTTCAAA GTTGTTTTCA AAGTTGGCGT ATAACATAGT ATCGACGGAG 10201 CCGATTTTGA AACCGCGGTG ATCACAGGCA GCAACGCTCT GTCATCGTTA 10251 CAATCAACAT GCTACCCTCC GCGAGATCAT CCGTGTTTCA AACCCGGCAG 10301 CTTAGTTGCC GTTCTTCCGA ATAGCATCGG TAACATGAGC AAAGTCTGCC 10351 GCCTTACAAC GGCTCTCCCG CTGACGCCGT CCCGGACTGA TGGGCTGCCT 10401 GTATCGAGTG GTGATTTTGT GCCGAGCTGC CGGTCGGGGA GCTGTTGGCT 10451 GGCTGGTGGC AGGATATATT GTGGTGTAAA CAAATTGACG CTTAGACAAC 10501 TTAATAACAC ATTGCGGACG TTTTTAATGT ACTGAATTAA CGCCGAATTA 10551 ATTCCTAGGC CACCATGTTG GGCCCGGGGC GCGCCGTACG TAGTGTTTAT 10601 CTTTGTTGCT TTTCTGAACA ATTTATTTAC TATGTAAATA TATTATCAAT 10651 GTTTAATCTA TTTTAATTTG CACATGAATT TTCATTTTAT TTTTACTTTA 10701 CAAAACAAAT AAATATATAT GCAAAAAAAT TTACAAACGA TGCACGGGTT 10751 ACAAACTAAT TTCATTAAAT GCTAATGCAG ATTTTGTGAA GTAAAACTCC 10801 AATTATGATG AAAAATACCA CCAACACCAC CTGCGAAACT GTATCCCAAC 10851 TGTCCTTAAT AAAAATGTTA AAAAGTATAT TATTCTCATT TGTCTGTCAT 10901 AATTTATGTA CCCCACTTTA ATTTTTCTGA TGTACTAAAC CGAGGGCAAA 10951 CTGAAACCTG TTCCTCATGC AAAGCCCCTA CTCACCATGT ATCATGTACG 11001 TGTCATCACC CAACAACTCC ACTTTTGCTA TATAACAACA CCCCCGTCAC 11051 ACTCTCCCTC TCTAACACAC ACCCCACTAA CAATTCCTTC ACTTGCAGCA 11101 CTGTTGCATC ATCATCTTCA TTGCAAAACC CTAAACTTCA CCTTCAACCG 11151 CGGCCGCATG GCTTCTATGA TATCCTCTTC CGCTGTGACA ACAGTCAGCC 11201 GTGCCTCTAG GGGGCAATCC GCCGCAGTGG CTCCATTCGG CGGCCTCAAA 11251 TCCATGACTG GATTCCCAGT GAAGAAGGTC AACACTGACA TTACTTCCAT 11301 TACAAGCAAT GGTGGAAGAG TAAAGTGCAT GCAGGTGTGG CCTCCAATTG 11351 GAAAGAAGAA GTTTGAGACT CTTTCCTATT TGCCACCATT GACGAGAGAT 11401 TCTAGAGTGA GTAACAAGAA CAACGATGAG CTGCAGTGGC AATCCTGGTT 11451 CAGCAAGGCG CCCACCACCG AGGCGAACCC GATGGCCACC ATGTTGCAGG 11501 ATATCGGCGT TGCGCTCAAA CCGGAAGCGA TGGAGCAGCT GAAAAACGAT 11551 TATCTGCGTG ACTTCACCGC GTTGTGGCAG GATTTTTTGG CTGGCAAGGC 11601 GCCAGCCGTC AGCGACCGCC GCTTCAGCTC GGCAGCCTGG CAGGGCAATC 11651 CGATGTCGGC CATCATGTCC GCATCTTACC TGCTCAACGC CAAATTCCTC 11701 AGTGCCATGG TGGAGGCGGT GGACACCGCA CCCCAGCAAA AGCAGAAAAT 11751 ACGCTTTGCC GTGCAGCAGG TGATTGATGC CATGTCGCCC GCGAACTTCC 11801 TCGCCACCAA CCCGGAAGCG CAGCAAAAAC TGATTGAAAC CAAGGGCGAG 11851 AGCCTGACGC GTGGCCTGGT CAATATGCTG GGCGATATCA ACAAGGGCCA 11901 TATCTCGCTG TCGGACGAAT CGGCCTTTGA AGTGGGCCGC AACCTGGCCA 11951 TTACCCCGGG CACCGTGATT TACGAAAATC CGCTGTTCCA GCTGATCCAG 12001 TACACGCCGA CCACGCCGAC GGTCAGCCAG CGCCCGCTGT TGATGGTGCC 12051 GCCGTGCATC AACAAGTTCT ACATCCTCGA CCTTCAACCG GAAAATTCGC 12101 TGGTGCGCTA CGCGGTGGAG CAGGGCAACA CCGTGTTCCT GATCTCGTGG 12151 AGCAATCCGG ACAAGTCGCT GGCCGGCACC ACCTGGGACG ACTACGTGGA 12201 GCAGGGCGTG ATCGAAGCGA TCCGCATCGT CCAGGACGTC AGCGGCCAGG 12251 ACAAGCTGAA CATGTTCGGC TTCTGCGTGG GCGGCACCAT CGTTGCCACC 12301 GCACTGGCGG TACTGGCGGC GCGTGGCCAG CACCCGGCGG CCAGCCTGAC 12351 CCTGCTGACC ACCTTCCTCG ACTTCAGCGA CACCGGCGTG CTCGACGTCT 12401 TCGTCGATGA AACCCAGGTC GCGCTGCGTG AACAGCAATT GCGCGATGGC 12451 GGCCTGATGC CGGGCCGTGA CCTGGCCTCG ACCTTCTCGA GCCTGCGTCC 12501 GAACGACCTG GTATGGAACT ATGTGCAGTC GAACTACCTC AAAGGCAATG 12551 AGCCGGCGGC GTTTGACCTG CTGTTCTGGA ATTCGGACAG CACCAATTTG 12601 CCGGGCCCGA TGTTCTGCTG GTACCTGCGC AACACCTACC TGGAAAACAG 12651 CCTGAAAGTG CCGGGCAAGC TGACGGTGGC CGGCGAAAAG ATCGACCTCG 12701 GCCTGATCGA CGCCCCGGCC TTCATCTACG GTTCGCGCGA AGACCACATC 12751 GTGCCGTGGA TGTCGGCGTA CGGTTCGCTC GACATCCTCA ACCAGGGCAA 12801 GCCGGGCGCC AACCGCTTCG TGCTGGGCGC GTCCGGCCAT ATCGCCGGCG 12851 TGATCAACTC GGTGGCCAAG AACAAGCGCA GCTACTGGAT CAACGACGGT 12901 GGCGCCGCCG ATGCCCAGGC CTGGTTCGAT GGCGCGCAGG AAGTGCCGGG 12951 CAGCTGGTGG CCGCAATGGG CCGGGTTCCT GACCCAGCAT GGCGGCAAGA 13001 AGGTCAAGCC CAAGGCCAAG CCCGGCAACG CCCGCTACAC CGCGATCGAG 13051 GCGGCGCCCG GCCGTTACGT CAAAGCCAAG GGCTGAGCGG CCGCTGAGTA 13101 ATTCTGATAT TAGAGGGAGC ATTAATGTGT TGTTGTGATG TGGTTTATAT 13151 GGGGAAATTA AATAAATGAT GTATGTACCT CTTGCCTATG TAGGTTTGTG 13201 TGTTTTGTTT TGTTGTCTAG CTTTGGTTAT TAAGTAGTAG GGACGTTCGT 13251 TCGTGTCTCA AAAAAAGGGG TACTACCACT CTGTAGTGTA TATGGATGCT 13301 GGAAATCAAT GTGTTTTGTA TTTGTTCACC TCCATTGTTG AATTCAATGT 13351 CAAATGTGTT TTGCGTTGGT TATGTGTAAA ATTACTATCT TTCTCGTCCG 13401 ATGATCAAAG TTTTAAGCAA CAAAACCAAG GGTGAAATTT AAACTGTGCT 13451 TTGTTGAAGA TTCTTTTATC ATATTGAAAA TCAAATTACT AGCAGCAGAT 13501 TTTACCTAGC ATGAAATTTT ATCAACAGTA CAGCACTCAC TAACCAAGTT 13551 CCAAACTAAG ATGCGCCATT AACATCAGCC AATAGGCATT TTCAGCAAGG 13601 CGCGCCCGCG CCGATGTATG TGACAACCCT CGGGATTGTT GATTTATTTC 13651 AAAACTAAGA GTTTTTGTCT TATTGTTCTC GTCTATTTTG GATATCAATC 13701 TTAGTTTTAT ATCTTTTCTA GTTCTCTACG TGTTAAATGT TCAACACACT 13751 AGCAATTTGG CCTGCCAGCG TATGGATTAT GGAACTATCA AGTCTGTGAC 13801 GCGCCGTACG TAGTGTTTAT CTTTGTTGCT TTTCTGAACA ATTTATTTAC 13851 TATGTAAATA TATTATCAAT GTTTAATCTA TTTTAATTTG CACATGAATT 13901 TTCATTTTAT TTTTACTTTA CAAAACAAAT AAATATATAT GCAAAAAAAT 13951 TTACAAACGA TGCACGGGTT ACAAACTAAT TTCATTAAAT GCTAATGCAG 14001 ATTTTGTGAA GTAAAACTCC AATTATGATG AAAAATACCA CCAACACCAC 14051 CTGCGAAACT GTATCCCAAC TGTCCTTAAT AAAAATGTTA AAAAGTATAT 14101 TATTCTCATT TGTCTGTCAT AATTTATGTA CCCCACTTTA ATTTTTCTGA 14151 TGTACTAAAC CGAGGGCAAA CTGAAACCTG TTCCTCATGC AAAGCCCCTA 14201 CTCACCATGT ATCATGTACG TGTCATCACC CAACAACTCC ACTTTTGCTA 14251 TATAACAACA CCCCCGTCAC ACTCTCCCTC TCTAACACAC ACCCCACTAA 14301 CAATTCCTTC ACTTGCAGCA CTGTTGCATC ATCATCTTCA TTGCAAAACC 14351 CTAAACTTCA CCTTCAACCG CGGCCGCATG GCTTCTATGA TATCCTCTTC 14401 CGCTGTGACA ACAGTCAGCC GTGCCTCTAG GGGGCAATCC GCCGCAGTGG 14451 CTCCATTCGG CGGCCTCAAA TCCATGACTG GATTCCCAGT GAAGAAGGTC 14501 AACACTGACA TTACTTCCAT TACAAGCAAT GGTGGAAGAG TAAAGTGCAT 14551 GCAGGTGTGG CCTCCAATTG GAAAGAAGAA GTTTGAGACT CTTTCCTATT 14601 TGCCACCATT GACGAGAGAT TCTAGAGTGA CTCAGCGCAT TGCGTATGTG 14651 ACCGGCGGCA TGGGTGGTAT CGGAACCGCC ATTTGCCAGC GGCTGGCCAA 14701 GGATGGCTTT CGTGTGGTGG CCGGTTGCGG CCCCAACTCG CCGCGCCGCG 14751 AAAAGTGGCT GGAGCAGCAG AAGGCCCTGG GCTTCGATTT CATTGCCTCG 14801 GAAGGCAATG TGGCTGACTG GGACTCGACC AAGACCGCAT TCGACAAGGT 14851 CAAGTCCGAG GTCGGCGAGG TTGATGTGCT GATCAACAAC GCCGGTATCA 14901 CCCGCGACGT GGTGTTCCGC AAGATGACCC GCGCCGACTG GGATGCGGTG 14951 ATCGACACCA ACCTGACCTC GCTGTTCAAC GTCACCAAGC AGGTGATCGA 15001 CGGCATGGCC GACCGTGGCT GGGGCCGCAT CGTCAACATC TCGTCGGTGA 15051 ACGGGCAGAA GGGCCAGTTC GGCCAGACCA ACTACTCCAC CGCCAAGGCC 15101 GGCCTGCATG GCTTCACCAT GGCACTGGCG CAGGAAGTGG CGACCAAGGG 15151 CGTGACCGTC AACACGGTCT CTCCGGGCTA TATCGCCACC GACATGGTCA 15201 AGGCGATCCG CCAGGACGTG CTCGACAAGA TCGTCGCGAC GATCCCGGTC 15251 AAGCGCCTGG GCCTGCCGGA AGAGATCGCC TCGATCTGCG CCTGGTTGTC 15301 GTCGGAGGAG TCCGGTTTCT CGACCGGCGC CGACTTCTCG CTCAACGGCG 15351 GCCTGCATAT GGGCTGAGCG GCCGCTGAGT AATTCTGATA TTAGAGGGAG 15401 CATTAATGTG TTGTTGTGAT GTGGTTTATA TGGGGAAATT AAATAAATGA 15451 TGTATGTACC TCTTGCCTAT GTAGGTTTGT GTGTTTTGTT TTGTTGTCTA 15501 GCTTTGGTTA TTAAGTAGTA GGGACGTTCG TTCGTGTCTC AAAAAAAGGG 15551 GTACTACCAC TCTGTAGTGT ATATGGATGC TGGAAATCAA TGTGTTTTGT 15601 ATTTGTTCAC CTCCATTGTT GAATTCAATG TCAAATGTGT TTTGCGTTGG 15651 TTATGTGTAA AATTACTATC TTTCTCGTCC GATGATCAAA GTTTTAAGCA 15701 ACAAAACCAA GGGTGAAATT TAAACTGTGC TTTGTTGAAG ATTCTTTTAT 15751 CATATTGAAA ATCAAATTAC TAGCAGCAGA TTTTACCTAG CATGAAATTT 15801 TATCAACAGT ACAGCACTCA CTAACCAAGT TCCAAACTAA GATGCGCCAT 15851 TAACATCAGC CAATAGGCAT TTTCAGCAAG GCGCGTAA pMBXS364 (SEQ ID NO: 2) 1 CATGCCAACC ACAGGGTTCC CCTCGGGATC AAAGTACTTT GATCCAACCC 51 CTCCGCTGCT ATAGTGCAGT CGGCTTCTGA CGTTCAGTGC AGCCGTCTTC 101 TGAAAACGAC ATGTCGCACA AGTCCTAAGT TACGCGACAG GCTGCCGCCC 151 TGCCCTTTTC CTGGCGTTTT CTTGTCGCGT GTTTTAGTCG CATAAAGTAG 201 AATACTTGCG ACTAGAACCG GAGACATTAC GCCATGAACA AGAGCGCCGC 251 CGCTGGCCTG CTGGGCTATG CCCGCGTCAG CACCGACGAC CAGGACTTGA 301 CCAACCAACG GGCCGAACTG CACGCGGCCG GCTGCACCAA GCTGTTTTCC 351 GAGAAGATCA CCGGCACCAG GCGCGACCGC CCGGAGCTGG CCAGGATGCT 401 TGACCACCTA CGCCCTGGCG ACGTTGTGAC AGTGACCAGG CTAGACCGCC 451 TGGCCCGCAG CACCCGCGAC CTACTGGACA TTGCCGAGCG CATCCAGGAG 501 GCCGGCGCGG GCCTGCGTAG CCTGGCAGAG CCGTGGGCCG ACACCACCAC 551 GCCGGCCGGC CGCATGGTGT TGACCGTGTT CGCCGGCATT GCCGAGTTCG 601 AGCGTTCCCT AATCATCGAC CGCACCCGGA GCGGGCGCGA GGCCGCCAAG 651 GCCCGAGGCG TGAAGTTTGG CCCCCGCCCT ACCCTCACCC CGGCACAGAT 701 CGCGCACGCC CGCGAGCTGA TCGACCAGGA AGGCCGCACC GTGAAAGAGG 751 CGGCTGCACT GCTTGGCGTG CATCGCTCGA CCCTGTACCG CGCACTTGAG 801 CGCAGCGAGG AAGTGACGCC CACCGAGGCC AGGCGGCGCG GTGCCTTCCG 851 TGAGGACGCA TTGACCGAGG CCGACGCCCT GGCGGCCGCC GAGAATGAAC 901 GCCAAGAGGA ACAAGCATGA AACCGCACCA GGACGGCCAG GACGAACCGT 951 TTTTCATTAC CGAAGAGATC GAGGCGGAGA TGATCGCGGC CGGGTACGTG 1001 TTCGAGCCGC CCGCGCACGT CTCAACCGTG CGGCTGCATG AAATCCTGGC 1051 CGGTTTGTCT GATGCCAAGC TGGCGGCCTG GCCGGCCAGC TTGGCCGCTG 1101 AAGAAACCGA GCGCCGCCGT CTAAAAAGGT GATGTGTATT TGAGTAAAAC 1151 AGCTTGCGTC ATGCGGTCGC TGCGTATATG ATGCGATGAG TAAATAAACA 1201 AATACGCAAG GGGAACGCAT GAAGGTTATC GCTGTACTTA ACCAGAAAGG 1251 CGGGTCAGGC AAGACGACCA TCGCAACCCA TCTAGCCCGC GCCCTGCAAC 1301 TCGCCGGGGC CGATGTTCTG TTAGTCGATT CCGATCCCCA GGGCAGTGCC 1351 CGCGATTGGG CGGCCGTGCG GGAAGATCAA CCGCTAACCG TTGTCGGCAT 1401 CGACCGCCCG ACGATTGACC GCGACGTGAA GGCCATCGGC CGGCGCGACT 1451 TCGTAGTGAT CGACGGAGCG CCCCAGGCGG CGGACTTGGC TGTGTCCGCG 1501 ATCAAGGCAG CCGACTTCGT GCTGATTCCG GTGCAGCCAA GCCCTTACGA 1551 CATATGGGCC ACCGCCGACC TGGTGGAGCT GGTTAAGCAG CGCATTGAGG 1601 TCACGGATGG AAGGCTACAA GCGGCCTTTG TCGTGTCGCG GGCGATCAAA 1651 GGCACGCGCA TCGGCGGTGA GGTTGCCGAG GCGCTGGCCG GGTACGAGCT 1701 GCCCATTCTT GAGTCCCGTA TCACGCAGCG CGTGAGCTAC CCAGGCACTG 1751 CCGCCGCCGG CACAACCGTT CTTGAATCAG AACCCGAGGG CGACGCTGCC 1801 CGCGAGGTCC AGGCGCTGGC CGCTGAAATT AAATCAAAAC TCATTTGAGT 1851 TAATGAGGTA AAGAGAAAAT GAGCAAAAGC ACAAACACGC TAAGTGCCGG 1901 CCGTCCGAGC GCACGCAGCA GCAAGGCTGC AACGTTGGCC AGCCTGGCAG 1951 ACACGCCAGC CATGAAGCGG GTCAACTTTC AGTTGCCGGC GGAGGATCAC 2001 ACCAAGCTGA AGATGTACGC GGTACGCCAA GGCAAGACCA TTACCGAGCT 2051 GCTATCTGAA TACATCGCGC AGCTACCAGA GTAAATGAGC AAATGAATAA 2101 ATGAGTAGAT GAATTTTAGC GGCTAAAGGA GGCGGCATGG AAAATCAAGA 2151 ACAACCAGGC ACCGACGCCG TGGAATGCCC CATGTGTGGA GGAACGGGCG 2201 GTTGGCCAGG CGTAAGCGGC TGGGTTGTCT GCCGGCCCTG CAATGGCACT 2251 GGAACCCCCA AGCCCGAGGA ATCGGCGTGA CGGTCGCAAA CCATCCGGCC 2301 CGGTACAAAT CGGCGCGGCG CTGGGTGATG ACCTGGTGGA GAAGTTGAAG 2351 GCCGCGCAGG CCGCCCAGCG GCAACGCATC GAGGCAGAAG CACGCCCCGG 2401 TGAATCGTGG CACGCGGCCG CTGATCGAAT CCGCAAAGAA TCCCGGCAAC 2451 CGCCGGCAGC CGGTGCGCCG TCGATTAGGA AGCCGCCCAA GGGCGACGAG 2501 CAACCAGATT TTTTCGTTCC GATGCTCTAT GACGTGGGCA CCCGCGTCAG 2551 TCGCAGCATC ATGGACGTGG CCGTTTTCCG TCTGTCGAAG CGTGACCGAC 2601 GAGCTGGCGA GGTGATCCGC TACGAGCTTC CAGACGGGCA CGTAGAGGTT 2651 TCCGCAGGGC CGGCCGGCAT GGCCAGTGTG TGGGATTACG ACCTGGTACT 2701 GATGGCGGTT TCCCATCTAA CCGAATCCAT GAACCGATAC CGGGAAGGGA 2751 AGGGAGACAA GCCCGGCCGC GTGTTCCGTC CACACGTTGC GGACGTACTC 2801 AAGTTCTGCC GGCGAGCCGA TGGCGGAAAG CAGAAAGACG ACCTGGTAGA 2851 AACCTGCATT CGGTTAAACA CCACGCACGT TGCCATGCAG CGTACGAAGA 2901 AGGCCAAGAA CGGCCGCCTG GTGACGGTAT CCGAGGGTGA AGCCTTGATT 2951 AGCCGCTACA AGATCGTAAA GAGCGAAACC GGGCGGCCGG AGTACATCGA 3001 GATCGAGCTA GCTGATTGGA TGTACCGCGA GATCACAGAA GGCAAGAACC 3051 CGGACGTGCT GACGGTTCAC CCCGATTACT TTTTGATCGA TCCCGGCATC 3101 GGCCGTTTTC TCTACCGCCT GGCACGCCGC GCCGCAGGCA AGGCAGAAGC 3151 CAGATGGTTG TTCAAGACGA TCTACGAACG CAGTGGCAGC GCCGGAGAGT 3201 TCAAGAAGTT CTGTTTCACC GTGCGCAAGC TGATCGGGTC AAATGACCTG 3251 CCGGAGTACG ATTTGAAGGA GGAGGCGGGG CAGGCTGGCC CGATCCTAGT 3301 CATGCGCTAC CGCAACCTGA TCGAGGGCGA AGCATCCGCC GGTTCCTAAT 3351 GTACGGAGCA GATGCTAGGG CAAATTGCCC TAGCAGGGGA AAAAGGTCGA 3401 AAAGGTCTCT TTCCTGTGGA TAGCACGTAC ATTGGGAACC CAAAGCCGTA 3451 CATTGGGAAC CGGAACCCGT ACATTGGGAA CCCAAAGCCG TACATTGGGA 3501 ACCGGTCACA CATGTAAGTG ACTGATATAA AAGAGAAAAA AGGCGATTTT 3551 TCCGCCTAAA ACTCTTTAAA ACTTATTAAA ACTCTTAAAA CCCGCCTGGC 3601 CTGTGCATAA CTGTCTGGCC AGCCCACAGC CGAAGAGCTG CAAAAAGCGC 3651 CTACCCTTCG GTCGCTGCGC TCCCTACGCC CCGCCGCTTC GCGTCGGCCT 3701 ATCGCGGCCG CTGGCCGCTC AAAAATGGCT GGCCTACGGC CAGGCAATCT 3751 ACCAGGGCGC GGACAAGCCG CGCCGTCGCC ACTCGACCGC CGGCGCCCAC 3801 ATCAAGGCAC CCTGCCTCGC GCGTTTCGGT GATGACGGTG AAAACCTCTG 3851 ACACATGCAG CTCCCGGAGA CGGTCACAGC TTGTCTGTAA GCGGATGCCG 3901 GGAGCAGACA AGCCCGTCAG GGCGCGTCAG CGGGTGTTGG CGGGTGTCGG 3951 GGCGCAGCCA TGACCCAGTC ACGTAGCGAT AGCGGAGTGT ATACTGGCTT 4001 AACTATGCGG CATCAGAGCA GATTGTACTG AGAGTGCACC ATATGCGGTG 4051 TGAAATACCG CACAGATGCG TAAGGAGAAA ATACCGCATC AGGCGCTCTT 4101 CCGCTTCCTC GCTCACTGAC TCGCTGCGCT CGGTCGTTCG GCTGCGGCGA 4151 GCGGTATCAG CTCACTCAAA GGCGGTAATA CGGTTATCCA CAGAATCAGG 4201 GGATAACGCA GGAAAGAACA TGTGAGCAAA AGGCCAGCAA AAGGCCAGGA 4251 ACCGTAAAAA GGCCGCGTTG CTGGCGTTTT TCCATAGGCT CCGCCCCCCT 4301 GACGAGCATC ACAAAAATCG ACGCTCAAGT CAGAGGTGGC GAAACCCGAC 4351 AGGACTATAA AGATACCAGG CGTTTCCCCC TGGAAGCTCC CTCGTGCGCT 4401 CTCCTGTTCC GACCCTGCCG CTTACCGGAT ACCTGTCCGC CTTTCTCCCT 4451 TCGGGAAGCG TGGCGCTTTC TCATAGCTCA CGCTGTAGGT ATCTCAGTTC 4501 GGTGTAGGTC GTTCGCTCCA AGCTGGGCTG TGTGCACGAA CCCCCCGTTC 4551 AGCCCGACCG CTGCGCCTTA TCCGGTAACT ATCGTCTTGA GTCCAACCCG 4601 GTAAGACACG ACTTATCGCC ACTGGCAGCA GCCACTGGTA ACAGGATTAG 4651 CAGAGCGAGG TATGTAGGCG GTGCTACAGA GTTCTTGAAG TGGTGGCCTA 4701 ACTACGGCTA CACTAGAAGG ACAGTATTTG GTATCTGCGC TCTGCTGAAG 4751 CCAGTTACCT TCGGAAAAAG AGTTGGTAGC TCTTGATCCG GCAAACAAAC 4801 CACCGCTGGT AGCGGTGGTT TTTTTGTTTG CAAGCAGCAG ATTACGCGCA 4851 GAAAAAAAGG ATCTCAAGAA GATCCTTTGA TCTTTTCTAC GGGGTCTGAC 4901 GCTCAGTGGA ACGAAAACTC ACGTTAAGGG ATTTTGGTCA TGCATTCTAG 4951 GTACTAAAAC AATTCATCCA GTAAAATATA ATATTTTATT TTCTCCCAAT 5001 CAGGCTTGAT CCCCAGTAAG TCAAAAAATA GCTCGACATA CTGTTCTTCC 5051 CCGATATCCT CCCTGATCGA CCGGACGCAG AAGGCAATGT CATACCACTT 5101 GTCCGCCCTG CCGCTTCTCC CAAGATCAAT AAAGCCACTT ACTTTGCCAT 5151 CTTTCACAAA GATGTTGCTG TCTCCCAGGT CGCCGTGGGA AAAGACAAGT 5201 TCCTCTTCGG GCTTTTCCGT CTTTAAAAAA TCATACAGCT CGCGCGGATC 5251 TTTAAATGGA GTGTCTTCTT CCCAGTTTTC GCAATCCACA TCGGCCAGAT 5301 CGTTATTCAG TAAGTAATCC AATTCGGCTA AGCGGCTGTC TAAGCTATTC 5351 GTATAGGGAC AATCCGATAT GTCGATGGAG TGAAAGAGCC TGATGCACTC 5401 CGCATACAGC TCGATAATCT TTTCAGGGCT TTGTTCATCT TCATACTCTT 5451 CCGAGCAAAG GACGCCATCG GCCTCACTCA TGAGCAGATT GCTCCAGCCA 5501 TCATGCCGTT CAAAGTGCAG GACCTTTGGA ACAGGCAGCT TTCCTTCCAG 5551 CCATAGCATC ATGTCCTTTT CCCGTTCCAC ATCATAGGTG GTCCCTTTAT 5601 ACCGGCTGTC CGTCATTTTT AAATATAGGT TTTCATTTTC TCCCACCAGC 5651 TTATATACCT TAGCAGGAGA CATTCCTTCC GTATCTTTTA CGCAGCGGTA 5701 TTTTTCGATC AGTTTTTTCA ATTCCGGTGA TATTCTCATT TTAGCCATTT 5751 ATTATTTCCT TCCTCTTTTC TACAGTATTT AAAGATACCC CAAGAAGCTA 5801 ATTATAACAA GACGAACTCC AATTCACTGT TCCTTGCATT CTAAAACCTT 5851 AAATACCAGA AAACAGCTTT TTCAAAGTTG TTTTCAAAGT TGGCGTATAA 5901 CATAGTATCG ACGGAGCCGA TTTTGAAACC GCGGTGATCA CAGGCAGCAA 5951 CGCTCTGTCA TCGTTACAAT CAACATGCTA CCCTCCGCGA GATCATCCGT 6001 GTTTCAAACC CGGCAGCTTA GTTGCCGTTC TTCCGAATAG CATCGGTAAC 6051 ATGAGCAAAG TCTGCCGCCT TACAACGGCT CTCCCGCTGA CGCCGTCCCG 6101 GACTGATGGG CTGCCTGTAT CGAGTGGTGA TTTTGTGCCG AGCTGCCGGT 6151 CGGGGAGCTG TTGGCTGGCT GGTGGCAGGA TATATTGTGG TGTAAACAAA 6201 TTGACGCTTA GACAACTTAA TAACACATTG CGGACGTTTT TAATGTACTG 6251 AATTAACGCC GAATTAATTC GGGGGATCTG GATTTTAGTA CTGGATTTTG 6301 GTTTTAGGAA TTAGAAATTT TATTGATAGA AGTATTTTAC AAATACAAAT 6351 ACATACTAAG GGTTTCTTAT ATGCTCAACA CATGAGCGAA ACCCTATAGG 6401 AACCCTAATT CCCTTATCTG GGAACTACTC ACACATTTTT ATGGAGAAAC 6451 TCGAGTTAAC CCTGAGACTG TTGGACAGAG CTCATTGGTA CCAGGAACAG 6501 GTGGTGGCGG CCCTCGGTGC GCTCGTACTG CTCCACGATG GTGTAGTCCT 6551 CGTTGTGGGA GGTGATGTCC AGCTTGGCGT CCACGTAGTA GTAGCCGGGC 6601 AGCTGCACGG GCTTCTTGGC CATGTAGATG GACTTGAACT CCACCAGGTA 6651 GTGGCCGCCG TCCTTCAGCT TCAGGGCCTT GTGGGTCTCG CCCTTCAGCA 6701 CGCCGTCGCG GGGGTACAGG CGCTCGGTGG AGGCCTCCCA GCCCATGGTC 6751 TTCTTCTGCA TCACGGGGCC GTCGGAGGGG AAGTTCACGC CGATGAACTT 6801 CACCTTGTAG ATGAAGCAGC CGTCCTGCAG GGAGGAGTCC TGGGTCACGG 6851 TCGCCACGCC GCCGTCCTCG AAGTTCATCA CGCGCTCCCA CTTGAAGCCC 6901 TCGGGGAAGG ACAGCTTCTT GTAGTCGGGG ATGTCGGCGG GGTGCTTCAC 6951 GTACACCTTG GAGCCGTACT GGAACTGGGG GGACAGGATG TCCCAGGCGA 7001 AGGGCAGGGG GCCGCCCTTG GTCACCTTCA GCTTCACGGT GTTGTGGCCC 7051 TCGTAGGGGC GGCCCTCGCC CTCGCCCTCG ATCTCGAACT CGTGGCCGTT 7101 CACGGTGCCC TCCATGCGCA CCTTGAAGCG CATGAACTCG GTGATGACGT 7151 TCTCGGAGGA GGCCATTTTG GTAGACTCGA GAGAGATAGA TTTGTAGAGA 7201 GAGACTGGTG ATTTCAGCGT GTCCTCTCCA AATGAAATGA ACTTCCTTAT 7251 ATAGAGGAAG GTCTTGCGAA GGATAGTGGG ATTGTGCGTC ATCCCTTACG 7301 TCAGTGGAGA TATCACATCA ATCCACTTGC TTTGAAGACG TGGTTGGAAC 7351 GTCTTCTTTT TCCACGATGC TCCTCGTGGG TGGGGGTCCA TCTTTGGGAC 7401 CACTGTCGGC AGAGGCATCT TGAACGATAG CCTTTCCTTT ATCGCAATGA 7451 TGGCATTTGT AGGTGCCACC TTCCTTTTCT ACTGTCCTTT TGATGAAGTG 7501 ACAGATAGCT GGGCAATGGA ATCCGAGGAG GTTTCCCGAT ATTACCCTTT 7551 GTTGAAAAGT CTCAATAGCC CTTTGGTCTT CTGAGACTGT ATCTTTGATA 7601 TTCTTGGAGT AGACGAGAGT GTCGTGCTCC ACCATGTTAT CACATCAATC 7651 CACTTGCTTT GAAGACGTGG TTGGAACGTC TTCTTTTTCC ACGATGCTCC 7701 TCGTGGGTGG GGGTCCATCT TTGGGACCAC TGTCGGCAGA GGCATCTTGA 7751 ACGATAGCCT TTCCTTTATC GCAATGATGG CATTTGTAGG TGCCACCTTC 7801 CTTTTCTACT GTCCTTTTGA TGAAGTGACA GATAGCTGGG CAATGGAATC 7851 CGAGGAGGTT TCCCGATATT ACCCTTTGTT GAAAAGTCTC AATAGCCCTT 7901 TGGTCTTCTG AGACTGTATC TTTGATATTC TTGGAGTAGA CGAGAGTGTC 7951 GTGCTCCACC ATGTTGGCAA GCTGCTCTAG CCAATACGCA AACCGCCTCT 8001 CCCCGCGCGT TGGCCGATTC ATTAATGCAG CTGGCACGAC AGGTTTCCCG 8051 ACTGGAAAGC GGGCAGTGAG CGCAACGCAA TTAATGTGAG TTAGCTCACT 8101 CATTAGGCAC CCCAGGCTTT ACACTTTATG CTTCCGGCTC GTATGTTGTG 8151 TGGAATTGTG AGCGGATAAC AATTTCACAC AGGAAACAGC TATGACCATG 8201 ATTACGAATT CAGGTACCAT TTAAATCCTG CAGGGTTTAA ACAGTGTTTT 8251 ACTCCTCATA TTAACTTCGG TCATTAGAGG CCACGATTTG ACACATTTTT 8301 ACTCAAAACA AAATGTTTGC ATATCTCTTA TAATTTCAAA TTCAACACAC 8351 AACAAATAAG AGAAAAAACA AATAATATTA ATTTGAGAAT GAACAAAAGG 8401 ACCATATCAT TCATTAACTC TTCTCCATCC ATTTCCATTT CACAGTTCGA 8451 TAGCGAAAAC CGAATAAAAA ACACAGTAAA TTACAAGCAC AACAAATGGT 8501 ACAAGAAAAA CAGTTTTCCC AATGCCATAA TACTCGAACG GCGCGCCTCA 8551 GCCCATATGC AGGCCGCCGT TGAGCGAGAA GTCGGCGCCG GTCGAGAAAC 8601 CGGACTCCTC CGACGACAAC CAGGCGCAGA TCGAGGCGAT CTCTTCCGGC 8651 AGGCCCAGGC GCTTGACCGG GATCGTCGCG ACGATCTTGT CGAGCACGTC 8701 CTGGCGGATC GCCTTGACCA TGTCGGTGGC GATATAGCCC GGAGAGACCG 8751 TGTTGACGGT CACGCCCTTG GTCGCCACTT CCTGCGCCAG TGCCATGGTG 8801 AAGCCATGCA CGCCGGCCTT GGCGGTGGAG TAGTTGGTCT GGCCGAACTG 8851 GCCCTTCTGC CCGTTCACCG ACGAGATGTT GACGATGCGG CCCCAGCCAC 8901 GGTCGGCCAT GCCGTCGATC ACCTGCTTGG TGACGTTGAA CAGCGAGGTC 8951 AGGTTGGTGT CGATCACCGC ATCCCAGTCG GCGCGGGTCA TCTTGCGGAA 9001 CACCACGTCG CGGGTGATAC CGGCGTTGTT GATCAGCACA TCAACCTCGC 9051 CGACCTCGGA CTTGACCTTG TCGAATGCGG TCTTGGTCGA GTCCCAGTCA 9101 GCCACATTGC CTTCCGAGGC AATGAAATCG AAGCCCAGGG CCTTCTGCTG 9151 CTCCAGCCAC TTTTCGCGGC GCGGCGAGTT GGGGCCGCAA CCGGCCACCA 9201 CACGAAAGCC ATCCTTGGCC AGCCGCTGGC AAATGGCGGT TCCGATACCA 9251 CCCATGCCGC CGGTCACATA CGCAATGCGC TGAGTCACTC TAGAATCTCT 9301 CGTCAATGGT GGCAAATAGG AAAGAGTCTC AAACTTCTTC TTTCCAATTG 9351 GAGGCCACAC CTGCATGCAC TTTACTCTTC CACCATTGCT TGTAATGGAA 9401 GTAATGTCAG TGTTGACCTT CTTCACTGGG AATCCAGTCA TGGATTTGAG 9451 GCCGCCGAAT GGAGCCACTG CGGCGGATTG CCCCCTAGAG GCACGGCTGA 9501 CTGTTGTCAC AGCGGAAGAG GATATCATAG AAGCCATTTT ACTAGTAAGA 9551 AGCTGAAAAT ATCAAAAGAA GGAACAGTCA TTAATCTATT GCATGTACTA 9601 GATTTTAGAT ATGAGTGGTC AAAAAAAACT TACGTTAATA ACGATGAAGA 9651 AGACAATGAT CCTCAGCACA ATCTCTCTCT CTCTCTCTTG GCTTCTCTTC 9701 TGGTGAATAG CACGAGAGAG GGTTTAAATG GAAGGCTCGT GGGTCCAAAA 9751 TGGGTGGCGG AGGAAATAGG AGAAGTAGGC AGTGACAAGT AATGTAGTAT 9801 TTAGTATTTG ATGAATGACA CATTTTCATT TCAGCATCAT CACCAACCAT 9851 CCTTTTGTTC CTTTGCTTCA ACTGTCACTT TCAATTGACA AAATTTTTTA 9901 TGTTTTCATG AGAAAACTAA ATTCTTATAA AGATTCATCT TCTTGAGTAT 9951 TATACGTGTA GTTTATGAAC AACACGTGTT GTTCCTATAT TTTTGTTCTG 10001 TTACCTCTAG AATAAAGTTG TCACCATTTC ATGAGTTCAA TTTTTCTTTA 10051 ATAGCCCCAA AAACAAAAGA TGATTCACAA GAAAGATGCG AATATTTTGC 10101 TATGAATCTT TTCTTAAGAG AAGCAATTAC ATTTTCACAA TAAAATTAGA 10151 TCCACGACTT AACCTAGTTT ATGTTGATTA TTTCTAGTGT TAGTATTAAG 10201 CAAAAATAAA ACTTATGAAT ACGAAGGCCT TTAAAGGAAA CTAAAGAAAG 10251 GACAAGGTAT AAACGTCCTA GAAAGTTCTA GGGTTTAGGC TTAGGGTCTA 10301 AGATATATGC TTTGAGTTTT ATGGCTTAGT AACACATTTT TGTAACACTT 10351 CTTTGTAACA TTTCTTGATA TGTTGGAGAA GTAACTCGTC TGGACAATAG 10401 TTATTTCCAA TATATAGGAA AAACGGCCTA AACAATAGCC GACGGGGACA 10451 AATACATCAT AAACAAAAAA TCCCGGTTAC AAACTTCCTA AAAAGCCATT 10501 CGGTCCACTC CGTTAAGCCT GAACTGTGCC TCCGTTATGC AAAAACGCCG 10551 TTGACCATCC GTAACCTAGT TGACTGACGG ATTATGGATT TAATCCGTTT 10601 TAAGGCCGTT AATAACACCA AAACGACGTC GTTTTGGTGT TTAAATTTTT 10651 TTTAACAACA ATTAAACCAA ACGACGTCGT TTTGGTTTAA TTAAATTTTT 10701 TTATCAAAAA CCCAAGCCCA AGCCCAAAAC TCTTAACAAA AGATAAAGCC 10751 CATCTCTATT TTTTCTAATT AAAACGCACA GCATTATGTT TCTTCTCTAA 10801 CGGATATATT TTCAATCTCA TAAATTGGGG ATTAGGGTTC TTATTTCCCA 10851 ATTCTCAATC TCTCAAAATT CTCCAAAATT CTCTGAAATT GATAATGCCT 10901 TCTTCTTCTT CAAACTCGTT TTTCTCTTTT GACAGTGAGC TTGAAGATGA 10951 TAACCATCGT GGTTTTCCTA AGACCTGTCG ATTTGGATGT CGTGTTGTGA 11001 TCAGAACCTC AAGAACTCCA AAAAACCTAG GTAGATTATT CCATACCTGT 11051 GAGAAAAATT TCAAAAGAGG AGGATTCCAC ACCTGGAAGT GGACTGATGT 11101 GTCTTTAGTA GAAGAAGTAG AGGACATAAA GGCTTACATT CATAACCGTG 11151 AGAAGTGTCA CGATGAAGAA ATGTTATTAT TGAAGGCTCA GATTCGTGGC 11201 TGTGAGAAGA TGATTGAAGG CTTGAAAGGA GAAGCAAAAC GTATGAAGCT 11251 AATTGTTGTT GCCGGAATAG TTGTGTTTGG TTGCTTTTTG TGTCTCTCTA 11301 AGTGATGTAT GAGATGAATG TTTGTGTATG TGATGTTGTT TTGTCTCAAT 11351 AATTAGTCAC TGATGTTGTA TGTAATGTTG TGTTTTGCAT CTCTAATTAG 11401 TTAATAATGA ATGTTGTTCT TATGTAATGT TTGATTTAAT CAATGGCTTT 11451 TGCAAATAAA TCCATAACAG AACNTATTCA ATATTTTCGA AAACATAACA 11501 AAGGTTTCAA AAGAAATTGC ATGTTGATTA GCTGAGTTTT CAAACAAAAT 11551 GCATTACATA GACAGACCCT GCTTCATAAT CCCCAAAACA CAAAAGAGAA 11601 GCATGCTAAT AACCGCAACT AATATCCAAA GACAGCTTCA TAATCCCAAA 11651 ACACAAAAAA AGAAGATTCA TAACCGATCC TTCATGTATT TAAAGAAAAT 11701 CAGACAACAA GCAAAGACTT AATCTTCCTG AGTAACTGAT GAGCTCAAGT 11751 CGACGTTTAA ACAGTGTTTT ACTCCTCATA TTAACTTCGG TCATTAGAGG 11801 CCACGATTTG ACACATTTTT ACTCAAAACA AAATGTTTGC ATATCTCTTA 11851 TAATTTCAAA TTCAACACAC AACAAATAAG AGAAAAAACA AATAATATTA 11901 ATTTGAGAAT GAACAAAAGG ACCATATCAT TCATTAACTC TTCTCCATCC 11951 ATTTCCATTT CACAGTTCGA TAGCGAAAAC CGAATAAAAA ACACAGTAAA 12001 TTACAAGCAC AACAAATGGT ACAAGAAAAA CAGTTTTCCC AATGCCATAA 12051 TACTCGAACT ACGTATTATT TGCGCTCGAC TGCCAGCGCC ACGCCCATGC 12101 CGCCGCCGAT GCACAGCGAG GCCAGGCCCT TCTTCGCGTC ACGGCGCTTC 12151 ATCTCGTGCA GCAGCGTCAC CAGGATACGG CAGCCCGACG CGCCGATCGG 12201 GTGGCCGATG GCGATGGCGC CGCCGTTCAC ATTGACCTTG GAGGTGTCCC 12251 AGCCCATCTG CTGGTGCACC GCCAGCGCCT GCGCGGCAAA GGCCTCGTTG 12301 ATCTCCATCA GGTCCAGGTC TTGCGGGGTC CACTCGGCGC GCGACAGGGC 12351 GCGCTTGGAG GCCGGCACCG GGCCCATGCC CATCACCTTG GGATCGACAC 12401 CGGCGTTGGC ATAGCTCTTG ATCGTGGCCA GCGGGGTCAG GCCCAGTTCC 12451 TTGGCCTTGG CCGCCGACAT CACCACCACC GCGGCGGCGC CGTCGTTCAG 12501 GCCCGAGGCG TTGGCCGCGG TCACCGTGCC GGCCTTGTCG AAGGCGGGCT 12551 TGAGGCCGGA CATGCTGTCC AGCGTGGCGC CCTGGCGCAC GAACTCGTCG 12601 GTCTTGAAGG CCACCGGGTC GCCCTTGCGC TGCGGGATCA GCACCGGGAC 12651 GATCTCTTCG TCAAACTTGC CGGCCTTCTG CGCGGCTTCG GCCTTGTTCT 12701 GCGAGCCGAC GGCGAACTCA TCCTGCGCCT CGCGTGTGAT GCCGTATTCC 12751 TTGGCCACGT TCTCGGCGGT GATGCCCATG TGGTACTGGT TGTACACGTC 12801 CCACAGGCCG TCGACGATCA TGGTGTCGAC CAGCTTGGCA TCGCCCATGC 12851 GGAAACCATC GCGCGAGCCC GGCAGCACGT GCGGGGCGGC GCTCATGTTT 12901 TCCTGGCCGC CGGCCACCAC GATCTCGGCG TCGCCCGCCA TGATCGCGTT 12951 GGCGGCCAGC ATCACGGCCT TCAGGCCCGA GCCGCACACC TTGTTGATGG 13001 TCATGGCCGG CACCATCGCC GGCAGGCCGG CCTTGATCGC GGCCTGGCGT 13051 GCGGGGTTCT GGCCCGAACC GGCGGTCAGC ACCTGGCCCA TGATGACTTC 13101 GCTCACCTGC TCCGGCTTGA CGCCGGCGCG CGCCGGCGCG GCCTTGATGA 13151 CCACGGCACC CAGTTCCGGT GCGGGGTTCT TGGCCAGCGA GCCGCCAAAC 13201 TTGCCGACCG CGGTGCGGGC GGCGGATACG ATGACAACGT CAGTCACTCT 13251 AGAATCTCTC GTCAATGGTG GCAAATAGGA AAGAGTCTCA AACTTCTTCT 13301 TTCCAATTGG AGGCCACACC TGCATGCACT TTACTCTTCC ACCATTGCTT 13351 GTAATGGAAG TAATGTCAGT GTTGACCTTC TTCACTGGGA ATCCAGTCAT 13401 GGATTTGAGG CCGCCGAATG GAGCCACTGC GGCGGATTGC CCCCTAGAGG 13451 CACGGCTGAC TGTTGTCACA GCCGAACAGG ATATCATAGA AGCCATTTTG 13501 GATCCAAGAA GCTGAAAATA TCAAAAGAAG GAACAGTCAT TAATCTATTG 13551 CATGTACTAG ATTTTAGATA TGAGTGGTCA AAAAAAACTT ACGTTAATAA 13601 CGATGAAGAA GACAATGATC CTCAGCACAA TCTCTCTCTC TCTCTCTTGG 13651 CTTCTCTTCT GGTGAATAGC ACGAGAGAGG GTTTAAATGG AAGGCTCGTG 13701 GGTCCAAAAT GGGTGGCGGA GGAAATAGGA GAAGTAGGCA GTGACAAGTA 13751 ATGTAGTATT TAGTATTTGA TGAATGACAC ATTTTCATTT CAGCATCATC 13801 ACCAACCATC CTTTTGTTCC TTTGCTTCAA CTGTCACTTT CAATTGACAA 13851 AATTTTTTAT GTTTTCATGA GAAAACTAAA TTCTTATAAA GATTCATCTT 13901 CTTGAGTATT ATACGTGTAG TTTATGAACA ACACGTGTTG TTCCTATATT 13951 TTTGTTCTGT TACCTCTAGA ATAAAGTTGT CACCATTTCA TGAGTTCAAT 14001 TTTTCTTTAA TAGCCCCAAA AACAAAAGAT GATTCACAAG AAAGATGCGA 14051 ATATTTTGCT ATGAATCTTT TCTTAAGAGA AGCAATTACA TTTTCACAAT 14101 AAAATTAGAT CCACGACTTA ACCTAGTTTA TGTTGATTAT TTCTAGTGTT 14151 AGTATTAAGC ACAAATAAGA CTTATGAATA CGAAGGCCTT TAAAGGAAAC 14201 TAAAGAAAGG ACAAGGTATA AACGTCCTAG AAAGTTCTAG GGTTTAGGCT 14251 TAGGGTCTAA GATATATGCT TTGAGTTTTA TGGCTTAGTA ACACATTTTT 14301 GTAACACTTC TTTGTAACAT TTCTTGATAT GTTGGAGAAG TAACTCGTCT 14351 GGACAATAGT TATTTCCAAT ATATAGGAAA AACTTCCTAA ACAATAGCCG 14401 ACGGGGACAA ATACATCATA AACAAAAGAT CCCGGTTACA AACTTCCTAA 14451 AAAGCCATTC GGTCCACTCC GTTAAGCCTG AACTGTGCCT CCGTTATGCA 14501 AAAACGCCGT TGACCATCCG TAACCTAGTT GACTGACGGA TTATGGATTT 14551 AATCCGTTTT AAGGCCGTTA ATAACACCAA AACGACGTCG TTTTGGTGTT 14601 TTAATTTTTT TTAACAACAA TTAAACCAAA CGACGTCGTT TTGGTTTAAT 14651 TAAATTTTTT TATCAAAAAC CCAAGCCCAA GCCCAAAACT CTTAACAAAA 14701 GATAAAGCCC ATCTCTATTT TTTCTAATTA AAACGCACAG CATTATGTTT 14751 CTTCTCTAAC GGATATATTT TCAATCTCAT AAATTGGGGA TTAGGGTTCT 14801 TATTTCCCAA TTCTCAATCT CTCAAAATTC TCCAAAATTC TCTGAAATTG 14851 ATAATGCCTT CTTCTTCTTC AAACTCGTTT TTCTCTTTTG ACAGTGAGCT 14901 TGAAGATGAT AACCATCGTG GTTTTCCTAA GACCTGTCGA TTTGGATGTC 14951 GTGTTGTGAT CAGAACCTCA ATAACACCAA AAAACCTAGG TAGATTATTC 15001 CATACCTGTG AGAAAAATTT CAAAAGAGGA GGATTCCACA CCTGGAAGTG 15051 GACTGATGTG TCTTTAGTAG AAGAAGTAGA GGACATAAAG GCTTACATTC 15101 ATAACCGTGA GAAGTGTCAC GATGAAGAAA TGTTATTATT GAAGGCTCAG 15151 ATTCGTGGCT GTGAGAAGAT GATTGAAGGC TTGAAAGGAG AAGCAAAACG 15201 TATGAAGCTA ATTGTTCTTG CCGGAATAGT TGTGTTTGGT TGCTTTTTGT 15251 GTCTCTCTAA GTGATGTATG AGATGAATGT TTGTGTATGT GATGTTGTTT 15301 TGTCTCAATA ATTAGTCACT GATGTTGTAT GTAATGTTGT GTTTTGCATC 15351 TCTAATTAGT TAATAATGAA TGTTGTTCTT ATGTAATGTT TGATTTAATC 15401 AATGGCTTTT GCAAATAAAT CCATAACAGA ACNTATTCAA TATTTTCGAA 15451 AACATAACAA AGGTTTCAAA AGAAATTGCA TTAGCATTAG CTGAGTTTTC 15501 AAACAAAATG CATTACATAG ACAGACCCTG CTTCATAATC CCCAAAACAC 15551 AAAAGAGAAG CATGCTAATA ACCGCAACTA ATATCCAAAG ACAGCTTCAT 15601 AATCCCAAAA CACAAAAAAA GAAGATTCAT AACCGATCCT TCATGTATTT 15651 AAAGAAAATC AGACAACAAG CAAAGACTTA ATCTTCCTGA GTAATGGAAG 15701 AGCTCAACTG CAGGTTTAAA CAGTGTTTTA CTCCTCATAT TAACTTCGGT 15751 CATTAGAGGC CACGATTTGA CACATTTTTA CTCAAAACAA AATGTTTGCA 15801 TATCTCTTAT AATTTCAAAT TCAACACACA ACAAATAAGA GAAAAAACAA 15851 ATAATATTAA TTTGAGAATG AACAAAAGGA CCATATCATT CATTAACTCT 15901 TCTCCATCCA TTTCCATTTC ACAGTTCGAT AGCGAAAACC GAATAAAAAA 15951 CACAGTAAAT TACAAGCACA ACAAATGGTA CAAGAAAAAC AGTTTTCCCA 16001 ATGCCATAAT ACTCGAACGC GATCGCTCAG CCCTTGGCTT TGACGTAACG 16051 GCCGGGCGCC GCCTCGATCG CGGTGTAGCG GGCGTTGCCG GGCTTGGCCT 16101 TGGGCTTGAC CTTCTTGCCG CCATGCTGGG TCAGGAACCC GGCCCATTGC 16151 GGCCACCAGC TGCCCGGCAC TTCCTGCGCG CCATCGAACC AGGCCTGGGC 16201 ATCGGCGGCG CCACCGTCGT TGATCCAGTA GCTGCGCTTG TTCTTGGCCA 16251 CCGAGTTGAT CACGCCGGCG ATATGGCCGG ACGCGCCCAG CACGAAGCGG 16301 TTGGCGCCCG GCTTGCCCTG GTTGAGGATG TCGAGCGAAC CGTACGCCGA 16351 CATCCACGGC ACGATGTGGT CTTCGCGCGA ACCGTAGATG AAGGCCGGGG 16401 CGTCGATCAG GCCGAGGTCG ATCTTTTCGC CGGCCACCGT CAGCTTGCCC 16451 GGCACTTTCA GGCTGTTTTC CAGGTAGGTG TTGCGCAGGT ACCAGCAGAA 16501 CATCGGGCCC GGCAAATTGG TGCTGTCCGA ATTCCAGAAC AGCAGGTCAA 16551 ACGCCGCCGG CTCATTGCCT TTGAGGTAGT TCGACTGCAC ATAGTTCCAT 16601 ACCAGGTCGT TCGGACGCAG GCTCGAGAAG GTCGAGGCCA GGTCACGGCC 16651 CGGCATCAGG CCGCCATCGC GCAATTGCTG TTCACGCAGC GCGACCTGGG 16701 TTTCATCGAC GAAGACGTCG AGCACGCCGG TGTCGCTGAA GTCGAGGAAG 16751 GTGGTCAGCA GGGTCAGGCT GGCCGCCGGG TGCTGGCCAC GCGCCGCCAG 16801 TACCGCCAGT GCGGTGGCAA CGATGGTGCC GCCCACGCAG AAGCCGAACA 16851 TGTTCAGCTT GTCCTGGCCG CTGACGTCCT GGACGATGCG GATCGCTTCG 16901 ATCACGCCCT GCTCCACGTA GTCGTCCCAG GTGGTGCCGG CCAGCGACTT 16951 GTCCGGATTG CTCCACGAGA TCAGGAACAC GGTGTTGCCC TGCTCCACCG 17001 CGTAGCGCAC CAGCGAATTT TCCGGTTGCA GGTCGAGGAT GTAGAACTTG 17051 TTGATGCACG GCGGCACCAT CAACAGCGGG CGCTGGCTGA CCGTCGGCGT 17101 GGTCGGCGTG TACTGGATCA GCTGGAACAG CGGATTTTCG TAAATCACGG 17151 TGCCCGGGGT AATGGCCAGG TTGCGGCCCA CTTCAAAGGC CGATTCGTCC 17201 GACAGCGAGA TATGGCCCTT GTTGATATCG CCCAGCATAT TGACCAGGCC 17251 ACGCGTCAGG CTCTCGCCCT TGGTTTCAAT CAGTTTTTGC TGCGCTTCCG 17301 GGTTGGTGGC GAGGAAGTTC GCGGGCGACA TGGCATCAAT CACCTGCTGC 17351 ACGGCAAAGC GTATTTTCTG CTTTTGCTGG GGTGCGGTGT CCACCGCCTC 17401 CACCATGGCA CTGAGGAATT TGGCGTTGAG CAGGTAAGAT GCGGCATTGA 17451 AGGCCGACAT CGGATTGCCC TGCCAGGCTG CCGAGCTGAA GCGGCGGTCG 17501 CTGACGGCTG GCGCCTTGCC AGCCAAAAAA TCCTGCCACA ACGCGGTGAA 17551 GTCACGCAGA TAATCGTTTT TCAGCTGCTC CATCGCTTCC GGTTTGAGCG 17601 CAACGCCGAT ATCCTGCAAC ATGGTGGCCA TCGGGTTCGC CTCGGTGGTG 17651 GGCGCCTTGC TGAACCAGGA TTGCCACTGC AGCTCATCGT TGTTCTTGTT 17701 ACTCACTCTA GAATCTCTCG TCAATGGTGG CAAATAGGAA AGAGTCTCAA 17751 ACTTCTTCTT TCCAATTGGA GGCCACACCT GCATGCACTT TACTCTTCCA 17801 CCATTGCTTG TAATGGAAGT AATGTCAGTG TTGACCTTCT TCACTGGGAA 17851 TCCAGTCATG GATTTGAGGC CGCCGAATGG AGCCACTGCG GCGGATTGCC 17901 CCCTAGAGGC ACGGCTGACT GTTGTCACAG CGGAAGAGGA TATCATAGAA 17951 GCCATTTTTG TACAAAGAAG CTGAAAATAT CAAAAGAAGG AACAGTCATT 18001 AATCTATTGC ATGTACTAGA TTTTAGATAT GAGTGGTCAA AAAAAACTTA 18051 CGTTAATAAC GATGAAGAAG ACAATGATCC TCAGCACAAT CTCTCTCTCT 18101 CTCTCTTGGC TTCTCTTCTG GTGAATAGCA CGAGAGAGGG TTTAAATGGA 18151 AGGCTCGTGG GTCCAAAATG GGTGGCGGAG GAAATAGGAG AAGTAGGCAG 18201 TGACAAGTAA TGTAGTATTT AGTATTTGAT GAATGACACA TTTTCATTTC 18251 AGCATCATCA CCAACCATCC TTTTGTTCCT TTGCTTCAAC TGTCACTTTC 18301 AATTGACAAA ATTTTTTATG TTTTCATGAG AAAACTAAAT TCTTATAAAG 18351 ATTCATCTTC TTGAGTATTA TACGTGTAGT TTATGAACAA CACGTGTTGT 18401 TCCTATATTT TTGTTCTGTT ACCTCTAGAA TAAAGTTGTC ACCATTTCAT 18451 GAGTTCAATT TTTCTTTAAT AGCCCCAAAA ACAAAAGATG ATTCACAAGA 18501 AAGATGCGAA TATTTTGCTA TGAATCTTTT CTTAAGAGAA GCAATTACAT 18551 TTTCACAATA AAATTAGATC CACGACTTAA CCTAGTTTAT GTTGATTATT 18601 TCTAGTGTTA GTATTAAGCA AAAATAAAAC TTATGAATAC GAAGGCCTTT 18651 AAAGGAAACT AAAGAAAGGA CAAGGTATAA ACGTCCTAGA AAGTTCTAGG 18701 GTTTAGGCTT AGGGTCTAAG ATATATGCTT TGAGTTTTAT GGCTTAGTAA 18751 CACATTTTTG TAACACTTCT TTGTAACATT TCTTGATATG TTGGAGAAGT 18801 AACTCGTCTG GACAATAGTT ATTTCCAATA TATAGGAAAA ACGGCCTAAA 18851 CAATAGCCGA CGGGGACAAA TACATCATAA ACAAAAAATC CCGGTTACAA 18901 ACTTCCTAAA AAGCCATTCG GTCCACTCCG TTAAGCCTGA ACTGTGCCTC 18951 CGTTATGCAA AAACGCCGTT GACCATCCGT AACCTAGTTG ACTGACGGAT 19001 TATGGATTTA ATCCGTTTTA AGGCCGTTAA TAACACCAAA ACGACGTCGT 19051 TTTGGTGTTT TAATTTTTTT TAACAACAAT TAAACCAAAC GACGTCGTTT 19101 TGGTTTAATT AAATTTTTTT ATCAAAAACC CAAGCCCAAG CCCAAAACTC 19151 TTAACAAAAG ATAAAGCCCA TCTCTATTTT TTCTAATTAA AACGCACAGC 19201 ATTATGTTTC TTCTCTAACG GATATATTTT CAATCTCATA AATTGGGGAT 19251 TAGGGTTCTT ATTTCCCAAT TCTCAATCTC TAACACTTCT CCAAAATTCT 19301 CTGAAATTGA TAATGCCTTC TTCTTCTTCA AACTCGTTTT TCTCTTTTGA 19351 CAGTGAGCTT GAAGATGATA ACCATCGTGG TTTTCCTAAG ACCTGTCGAT 19401 TTGGATGTCG TGTTGTGATC AGAACCTCAA GAACTCCAAA AAACCTAGGT 19451 AGATTATTCC ATACCTGTGA GAAAAATTTC AAAAGAGGAG GATTCCACAC 19501 CTGGAAGTGG ACTGATGTGT CTTTAGTAGA AGAAGTAGAG GACATAAAGG 19551 CTTACATTCA TAACCGTGAG AAGTGTCACG ATGAAGAAAT GTTATTATTG 19601 AAGGCTCAGA TTCGTGGCTG TGAGAAGATG ATTGAAGGCT TGAAAGGAGA 19651 AGCAAAACGT ATGAAGCTAA TTGTTGTTGC CGGAATAGTT GTGTTTGGTT 19701 GCTTTTTGTG TCTCTCTAAG TGATGTATGA GATGAATGTT TGTGTATGTG 19751 ATGTTGTTTT GTCTCAATAA TTAGTCACTG ATGTTGTATG TAATGTTGTG 19801 TTTTGCATCT CTAATTAGTT AATAATGAAT GTTGTTCTTA TGTAATGTTT 19851 GATTTAATCA ATGGCTTTTG CAAATAAATC CATAACAGAA CNTATTCAAT 19901 ATTTTCGAAA ACATAACAAA GGTTTCAAAA GAAATTGCAT TAGCATTAGC 19951 TGAGTTTTCA AACAAAATGC ATTACATAGA CAGACCCTGC TTCATAATCC 20001 CCAAAACACA AAAGAGAAGC ATGCTAATAA CCGCAACTAA TATCCAAAGA 20051 CAGCTTCATA ATCCCAAAAC ACAAAAAAAG AAGATTCATA ACCGATCCTT 20101 CATGTATTTA AAGAAAATCA GACAACAAGC AAAGACTTAA TCTTCCTGAG 20151 TAACTGATGA GCTCAAAAGC TTGGCACTGG CCGTCGTTTT ACAACGTCGT 20201 GACTGGGAAA ACCCTGGCGT TACCCAACTT AATCGCCTTG CAGCACATCC 20251 CCCTTTCGCC AGCTGGCGTA ATAGCGAAGA GGCCCGCACC GATCGCCCTT 20301 CCCAACAGTT GCGCAGCCTG AATGGCGAAT GCTAGAGCAG CTTGAGCTTG 20351 GATCAGATTG TCGTTTCCCG CCTTCAGTTT AAACTATCAG TGTTTGACAG 20401 GATATATTGG CGGGTAAACC TAAGAGAAAA GAGCGTTTAT TAGAATAACG 20451 GATATTTAAA AGGGCGTGAA AAGGTTTATC CGTTCGTCCA TTTGTATGTG 

1. An oilseed comprising greater than 7% polyhydroxyalkanoate (PHA) dry weight of the oilseed, wherein germination of the oilseed is impaired relative to an oilseed having less than 7% polyhydroxyalkanoate.
 2. The oilseed of claim 1, wherein the PHA comprises (poly) 3-hydroxybutyrate (PHB).
 3. The oilseed of claim 1 comprises greater than 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% or 19% of the dry weight of the seed.
 4. The oilseed of claim 1, wherein the oilseed is produced by a transgenic plant genetically engineered to produce PHA.
 5. The oilseed of claim 4, wherein the PHA is PHB.
 6. The oilseed of claim 4, wherein the plant transformed to produce the transgenic plant is selected from the group consisting of members of the Brassica family: B. napus, B. rapa, B. carinata and B. juncea; industrial oilseeds: Camelina sativa, Crambe, jatropha, castor; Arabidopsis thaliana; Calendula, Cuphea; maize; soybean; cottonseed; sunflower; palm; coconut; safflower; peanut; mustards including Sinapis alba; and tobacco.
 7. The oilseed of claim 1, wherein germination of the oilseed is impaired by 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% relative to an oilseed comprising less than 7% PHA.
 8. A transgenic plant genetically engineered to produce PHA, wherein the transgenic plant produces the oilseed of claim
 1. 9. The transgenic plant of claim 8 wherein the plant transformed to produce the oilseed of claim 1 is selected from the group consisting of members of the Brassica family; B. napus, B. rapa, B. carinata and B. juncea; industrial oilseeds: Camelina sativa, Crambe, Jatropha, castor; Arabidopsis thaliana; Calendula, Cuphea; maize; soybean; cottonseed; sunflower; palm; coconut; safflower; peanut; mustards: Sinapis alba; and tobacco.
 10. (canceled)
 11. A method for producing a hybrid transgenic plant line comprising crossing a plant line comprising one or more PHB biosynthetic pathway genes with a plant line containing the remaining PHB biosynthetic pathway gene(s) needed to complete the PHB biosynthetic pathway.
 12. The method of claim 11 wherein the plant lines comprise cytoplasmic male sterility (CMS) controlled by an extranuclear genome.
 13. The method of claim 11 wherein the male sterile line is maintained by crossing with a maintainer line that is genetically identical and comprises normal fertile cytoplasm.
 14. The method of claim 13 wherein the maintainer line is transformed with one or more genes for the PHB biosynthetic pathway.
 15. The method of claim 14 wherein crossing the transformed maintainer line with the original male sterile line produces a male sterile line possessing a portion of the PHB biosynthetic pathway.
 16. The method of claim 15 wherein insertion of the phaA and phaC genes into the maintainer line and crossing with the original male cytoplasmic sterile line forms a male sterile line containing the phaA and phaC genes. 